Data Explorer™ Software Version 4 Series Software User Guide
© Copyright 2001, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication.
Table of Contents Table of Contents How to Use This Guide .............................................................. xi Chapter 1 Data Explorer™ Basics 1.1 1.2 .............................................................................. 1-2 File Formats and Types ............................................................ 1-5 Overview 1.2.1 Software Applications Compatibility ....................................... 1-5 1.2.2 Data (.DAT) File Format ............................................
Table of Contents Chapter 2 Using Chromatogram and Spectrum Windows 2.1 ................................................. 2-2 2.1.1 Opening Data Files ................................................................ 2-2 2.1.2 Displaying Mariner DAD Traces ............................................. 2-6 2.1.3 Displaying Voyager Chromatograms ...................................... 2-7 2.1.4 Viewing Read-Only Files ........................................................ 2-7 2.1.
Table of Contents Chapter 3 Peak Detection and Labeling 3.1 3.2 3.3 3.4 3.5 Overview .............................................................................. 3-2 3.1.1 Default Peak Detection .......................................................... 3-2 3.1.2 The Resolution-Based Peak Detection Routine ...................... 3-3 Peak Detection ....................................................................... 3-6 3.2.1 Strategy for Mariner Peak Detection ..............................
Table of Contents Chapter 4 Examining Chromatogram Data 4.1 4.2 .............................................................................. 4-2 Creating an Extracted Ion Chromatogram ..................................... 4-5 Overview 4.2.1 Creating an Extracted Ion Chromatogram (XIC) ..................... 4-5 4.2.2 Creating a Constant Neutral Loss (CNL) Chromatogram ........ 4-9 4.3 Creating an Extracted Absorbance Chromatogram (XAC) (Mariner Data Only) ...........................................
Table of Contents 5.5 5.6 5.7 5.8 .......................................................................... Mass Deconvolution (Mariner Data Only) ..................................... Noise Filtering/Smoothing ........................................................ Adjusting the Baseline ............................................................ Centroiding 5-36 5-37 5-42 5-45 5.8.1 Using Baseline Offset ...........................................................5-45 5.8.
Table of Contents Chapter 7 Data Explorer Examples 7.1 7.2 Mariner Data Examples ............................................................ 7-2 7.1.1 Improving Signal-To-Noise Ratio ............................................ 7-2 7.1.2 Deconvoluting and Evaluating Unresolved Chromatographic Peaks .......................................7-4 7.1.3 Determining if a Peak is Background Noise ............................ 7-8 Voyager Data Examples .......................................................
Table of Contents Appendix A Warranty Information ........................................ A-1 Appendix B Overview of Isotopes ........................................ B-1 Appendix C Data Explorer Toolbox (Visual Basic Macros) ......................................................... C-1 C.1 C.2 C.3 C.4 C.5 C.6 C.7 .............................................................................. C-2 Preparing Data Before Accessing Macros ..................................... C-3 Accessing the Macros ..
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How to Use This Guide 1 How to Use This Guide Purpose of this guide The Applied Biosystems Data Explorer ™ Software User’s Guide describes processing and analyzing data with the Data Explorer software. You can use the Data Explorer software to analyze data collected on: • Mariner™ Workstations with Version 3.0 and later software • Voyager™-DE Biospectrometry™ Workstations with Version 5.
1 How to Use This Guide Chapter/Appendix Content Chapter 5, Examining Spectrum Data Describes processing and analyzing mass spectral data. Chapter 6, Using Tools and Applications Describes how to generate results using several tools and applications: the Centroid calculator, Elemental Composition calculator, Isotope calculator, Mass Resolution calculator, Ion Fragmentation calculator and Signal-to-Noise calculator. Also describes using the Macro Recorder and the Elemental Targeting Application.
How to Use This Guide Conventions 1 This guide uses the following conventions to make text easier to understand. • Bold indicates user action. For example: Type 0 and press Enter for the remaining fields. • Italic text denotes new or important words, and is also used for emphasis. For example: Before analyzing, always prepare fresh matrix. Notes, Cautions, Warnings, and Hints A note provides important information to the operator.
1 How to Use This Guide Related documentation The related documents shipped with your system include: • Mariner ™ Workstation User’s Guide—Use this document to learn detailed information about the Mariner Workstation. • Voyager™-DE Biospectrometry Workstation User’s Guide—Use this document to learn detailed information about the Voyager-DE Workstation. • Printer documentation (depends on the printer you purchase)—Use this documentation to set up and service your printer.
Chapter Explorer™ 1 Data Basics 1 This chapter contains the following sections: 1.1 Overview ................................................... 1-2 1.2 File Formats and Types ............................. 1-5 1.3 Parts of the Data Explorer Window .......... 1-11 1.4 Customizing the Data Explorer Window ... 1-17 1.5 Setting Graphic Options .......................... 1-23 1.6 Managing Files ........................................
Chapter 1 Data Explorer™ Basics 1 1.1 Overview Description The Data Explorer™ Version 4.0 processing software is graphical software that you use to analyze, calibrate, and report data.
Overview Starting and exiting the software To start the Data Explorer software from the Windows NT desktop, double-click the Data Explorer icon on the desktop. The Data Explorer window opens. The Data Explorer window is blank with only a few menus displayed until you open a data file. Figure 1-1 shows the Data Explorer main window with a Mariner data file open. Figure 1-2 shows the Data Explorer main window with a Voyager data file open.
Chapter 1 Data Explorer™ Basics 1 Figure 1-2 Data Explorer Window with Voyager Data Default colors The default colors are different for Mariner and Voyager: • Mariner—Black background, yellow traces, and green labels • Voyager—White background, blue traces, and red labels You can customize the default colors as needed. See Section 1.5.1, Changing Background Color.
File Formats and Types 1.2 File Formats and Types 1 This section describes: • • Software applications compatibility Data (.DAT) file format 1.2.1 Software Applications Compatibility You can use the Data Explorer Macro Recorder function to create Visual Basic scripts to automate tasks. You can also use the Visual Basic Editor directly to create more complex programs customized to suit your needs. For more information, see Section 6.7, Using the Macro Recorder.
Chapter 1 1 Data Explorer™ Basics Mariner .SPC file format In Mariner software versions earlier than version 3.0, data files are stored in .SPC format. You can view and process .SPC files in Data Explorer, or you can convert these files to .DAT format. For information about the differences between the .DAT and the .SPC formats, see the Mariner™ Workstation User’s Guide. Voyager .MS file format In Voyager software versions earlier than version 5.0, data files are stored in .MS, .MSF, .MSA, and .
File Formats and Types Table 1-1 Information Stored In a .DAT File (Continued) Category Settings (continued) Display Process File Type 1 File Content .MSM (Mariner only) MS Method settings, if data was acquired using an .MSM file. .SET Graphic and processing settings. See Section 1.4.2, Customizing Processing and Graphic Settings (.SET). .LBC Chromatogram label information. See Section 3.5.3, Setting Custom Peak Labels. .LBS Spectrum label information. See Section 3.5.
Chapter 1 1 Data Explorer™ Basics Additional files types Additional file types you may see on your system are described below. Table 1-2 Additional File Types Category Data Data Procedure 1-8 File Type File Content .PKT Text file containing a chromatogram or a spectrum peak list that you can save from the Output window. See “Output window” on page 1-15. .TXT Data file exported to an ASCII text file. See Section 1.6.3, Converting to and Exporting ASCII Data. .
File Formats and Types Table 1-2 Additional File Types (Continued) Category File Type 1 File Content Reference .REF List of masses to select from during calibration. See “Creating and saving a calibration reference file” on page 5-18. Process .RCT (Mariner only) Results file saved from: • Mariner .SPC format data file (versions earlier than 3.0) in the Data Explorer software after a chromatogram is manually processed. See Section 2.8, Saving, Opening, and Deleting .
Chapter 1 Data Explorer™ Basics Table 1-2 Additional File Types (Continued) 1 Category Process File Type .RCD Chromatogram results file exported from .DAT files. See Section 2.7, Exporting, Opening, and Deleting .RCD and .RSD Results Files (Mariner Data Only). .RSD Spectrum results file exported from .DAT files. See Section 2.7, Exporting, Opening, and Deleting .RCD and .RSD Results Files (Mariner Data Only).
Parts of the Data Explorer Window 1.3 Parts of the Data Explorer Window 1 This section describes: • • • • • • Overview Overview Toolbar Chromatogram and Spectrum windows Tabs for data files Data file names Output window Figure 1-3 shows the Data Explorer window with Mariner data.
Chapter 1 Data Explorer™ Basics Toolbar 1 The toolbar contains buttons that access Data Explorer functions. For a description of a toolbar button, place the cursor on the button. A brief description of the button (ToolTip) is displayed below the button. For information on adding or removing toolbar buttons, see “Customizing toolbars” on page 1-21.
Parts of the Data Explorer Window Table 1-3 Mariner Data Displayed in Chromatogram and Spectrum Windows (Continued) Window SPEC Mariner Data Displays the spectrum for the selected time in the TIC or TAC trace. By default, displays spectrum #1. The trace label includes “DAD” for spectra selected from TAC. Indicates Base Peak (BP) mass and intensity for the tallest peak in the spectrum. Displayed as % Intensity versus Mass-to-Charge (m/z). The right axis is scaled to the intensity of the base peak.
Chapter 1 Data Explorer™ Basics Labels in the chromatogram or spectrum title identify the type of data displayed in the window. For a description of labels, see Section 2.4.10, Viewing Trace Labels. 1 For more information on Chromatogram and Spectrum windows, see Chapter 2, Using Chromatogram and Spectrum Windows.
Parts of the Data Explorer Window Output window Output window tabs The Output window (see Figure 1-3 on page 1-11) displays tabs at the bottom that you can click to switch between the types of information displayed: • Result—Displays results generated using commands on the Process, Tools, and Applications menus. For more information on results, see: • Section 5.3, Manual Calibration, or Section 5.4, Automatic Calibration • Section 5.6, Mass Deconvolution (Mariner Data Only) • Section 6.
Chapter 1 Data Explorer™ Basics 1 Displaying, clearing, and closing • Instrument Setting—Displays a list of instrument settings used to obtain the data. The settings are taken from instrument settings pages in the Instrument Control Panel. Also displays segments, event numbers, and event tags from Mariner MS Method acquisitions and LC information if LCMS was acquired using Mariner. • Elemental Analysis—Displays results for the Elemental Composition calculator. For information, see Section 6.
Customizing the Data Explorer Window 1.4 Customizing the Data Explorer Window 1 This section includes: • • • Setting default values Customizing Graphic and Processing settings Customizing toolbars 1.4.1 Setting Default Values You can set defaults for most dialog boxes in the Data Explorer software by setting a value or selecting a button, closing the dialog box, then closing the data file you are viewing. The next time you open the dialog box, the last settings specified are displayed. 1.4.
Chapter 1 1 Data Explorer™ Basics Overview of processing and graphic settings What settings contain Processing and graphic settings control how data is processed and displayed in the Data Explorer software. The last settings used are automatically saved in the data file when you close it. The next time you open the data file, you can select any of the following to apply: • Settings from the data file—Described in Section 2.1, Opening and Closing Data Files. • Default settings—Described below.
Customizing the Data Explorer Window Additional .SET files that have been developed for detection of different types of data are included in the C:\VOYAGER\PROGRAM\SET FILES directory. The names of the .SET files indicate the type of data the files can be used for. The appropriate default settings for the type of data you open are automatically applied to a data file the first time you open it in Data Explorer. You can also manually apply these settings if desired. For information, see “Applying a .
Chapter 1 1 Data Explorer™ Basics Opening, customizing, and saving .SET files To open, customize, and save .SET files: 1. If you are customizing a default .SET file, make a copy of the original file before opening it. See “Making a copy of default .SET files before customizing” on page 1-19. 2. Select Settings from the File Menu. 3. Select one of the following: • Restore Processing Settings • Restore Graphic Settings • Restore Graphic/Processing Settings 4.
Customizing the Data Explorer Window To use the Settings option: 1. Select Settings from the File menu. 2. Select one of the following: 1 • Restore Processing Settings • Restore Graphic Settings • Restore Graphic/Processing Settings • Revert to the Last Saved Graphic/Processing Settings 3. If you select a Restore Settings option, select or type the name of the .SET file in the Restore dialog box, then click OK.
Chapter 1 Data Explorer™ Basics 4. 1 To remove a button from a toolbar, click-drag the button from the toolbar. NOTE: The Customize dialog box must be displayed to click-drag a button from a toolbar. 5. Undocking toolbars Click OK to close the Customize dialog box. The toolbar at the top of the Data Explorer window is divided into sections. A section is preceded by a double vertical bar.
Setting Graphic Options 1.5 Setting Graphic Options 1 This section includes: • • • Changing background color Customizing options Reverting to previous graphic options NOTE: Changes you make to Graphic Options are saved with the data file. 1.5.1 Changing Background Color White or dark background You can switch background color by selecting Default from the Display menu, then selecting: • White Background—Displays blue traces and red labels by default. Default settings are contained in DEFAULTWHITE.
Chapter 1 1 Data Explorer™ Basics 1.5.2 Customizing Graphic Options This section includes: • • • • • • Accessing graphic options 1-24 Applied Biosystems Accessing graphic options Setting colors Setting line widths Setting data cursors Setting traces in line or bar mode Setting graphic compression To access graphic options: 1. Display the trace of interest. 2. From the Display menu, select Graphic Options. 3.
Setting Graphic Options 1 Line or vertical bar traces Peak bounds Line width Data cursor Graphic compression Figure 1-4 Graphic Options Dialog Box— Graph Setup Tab Setting colors Manually You can set colors manually or automatically. To manually select the color of graph features (axis, peak bounds, tick labels, data cursor) and plot features (traces, peak labels): 1. Select Graphic Options from the Display menu. 2. Set colors in the Graph Setup tab (see Figure 1-4).
Chapter 1 Data Explorer™ Basics When you manually set colors, note: 1 • Selections set to white (or line widths set to 0) may not print on certain printers. • If you select different trace colors for multiple traces, only the color for the active trace is saved when you close the data file. Automatically using Auto Color Automatically assigning trace colors is useful when overlaying traces. To automatically assign trace colors: 1. Select Graphic Options from the Display menu. 2.
Setting Graphic Options Setting data cursors To enable data cursors and set cursor labels and attributes: 1 1. Select Graphic Options from the Display menu. 2.
Chapter 1 1 Data Explorer™ Basics Setting traces in Line or Vertical Bar mode Setting graphic compression You can change the trace display from Line to Vertical Bars. Each vertical bar represents one data point. Vertical bar mode is useful when setting peak detection parameters to determine the number of points across a peak. NOTE: Graphic Compression mode is not saved as part of graphic settings. When you close a data file, it is automatically reset to the default Local Max setting.
Setting Graphic Options 1.5.3 Reverting to Previous Graphic Options You have two options to revert to previously used graphic options: • Revert to Last Saved Graphic Setting—Reverts to the last graphic settings saved in the data file. Does not affect processing settings. To access, select Default from the Display menu, then select Revert to Last Saved Graphic Setting. • Revert to Last Saved Graphic/Processing Settings—Reverts to the last graphic and processing settings saved in the data file.
Chapter 1 Data Explorer™ Basics 1 1.6 Managing Files This section describes: • Converting .SPC file format to .DAT file format (Mariner only) • Converting data from profile to centroid (Mariner only) • Converting to and exporting ASCII data • Importing a trace in ASCII format • Extracting and saving information from .DAT, .RSD, and .RCD files • Copying from data files 1.6.1 Converting .SPC File Format to .
Managing Files Before you begin Confirm that the .SPC and .CGM files are located in the same directory. Use Windows NT® Explorer to display the directory contents and to move the .SPC and .CGM files as necessary. If the .SPC and .CGM files are not in the same directory, when you open the .SPC file, a “Failed to open chromatogram data” message is displayed. To check that the .SPC and .CGM files are in the same directory: 1. Select Open from the File menu. The Open dialog box is displayed. 2.
Chapter 1 Data Explorer™ Basics The Convert to .DAT Format dialog box reappears. 1 5. Click OK. A message box is displayed, showing the file name of the newly created .DAT file. 6. Viewing file properties Close the .SPC file and open the .DAT file before processing. If desired, manually delete the .SPC file and associated files. File properties are accessible in Windows NT Explorer and provide general information about a data file without opening the file in the Data Explorer software.
Managing Files 1.6.2 Converting Data from Profile to Centroid (Mariner Data Only) Overview You can convert an entire data file from profile to centroid format. Centroid format files are smaller than profile format files. NOTE: Profile data is not automatically deleted when you convert to centroid data. You can delete the profile data file using Windows NT Explorer. Before you begin Converting to centroid If the file to convert is in .SPC format, confirm that the .SPC and .
Chapter 1 1 Data Explorer™ Basics 1.6.3 Converting to and Exporting ASCII Data This section describes: • • Converting a data file to ASCII format Converting a data file to ASCII format Exporting a trace to ASCII format You can convert a data file for use in a spreadsheet or another application and export and import single traces in ASCII format. To convert an entire active data file to an ASCII text file: 1. Open the data file to convert. 2.
Managing Files 1.6.4 Importing a Trace in ASCII Format 1 You can import trace data in ASCII format. If the file you are importing was originally exported using the Data Explorer software, you can import a spectrum trace only into the Spectrum window and a chromatogram trace only into the Chromatogram window. However, if the data is from another source, the software does not know the data type and you must make sure to import the data into the correct window type.
Chapter 1 Data Explorer™ Basics 1 CAUTION An imported ASCII format trace contains only the data points for the trace. The Sample Info and Instrument settings tabs in the Output window display data from the data file you opened in step 1. These tabs do not include information about the imported trace. 1.6.5 Extracting and Saving Information from .DAT, .RSD, and .RCD Files Overview You can extract the following information from a .DAT data file, .RSD spectrum results file, and .
Managing Files NOTE: To export calibration constants used to acquire the data, select Mass Calibration from the Process menu, then select the Revert to Instrument Calibration before exporting. For more information, see Section 5.3.4, Reverting to Instrument Calibration. • Configuration—To export .BIC or .MSM files (.MSM—Mariner data only). NOTE: To access the instrument settings for each spectrum in a data file acquired using a Mariner MS Method, you must first extract the .
Chapter 1 Data Explorer™ Basics Saving .LBS and .LBC files 1 To save spectrum (.LBS) or chromatogram (.LBC) peak label files from a .DAT, .RSD, or .RSC file, see Section 3.5.3, Setting Custom Peak Labels. 1.6.6 Copying from Data Files Overview Copy trace image 1-38 Applied Biosystems You can copy the following types of data from data and result files to the Windows clipboard: • Trace Image—Copies the graphic image of the trace in the active window.
Managing Files NOTE: If you paste the image into an application that does not handle Windows Metafile format images, for example Microsoft Paint, images are distorted. Copy trace data To copy raw data (x,y pairs) for the peaks displayed in the active trace to the Windows clipboard: 1. Select the trace window to copy. Zoom and adjust the trace as needed. NOTE: Only raw data for the set of peaks displayed in the trace window is copied. 2. From the Edit menu, select Copy, then select Trace Data. 3.
Chapter 1 Data Explorer™ Basics 1 NOTE: Copy Displayed Peaks copies all fields and headings. However, some data applications may not work correctly if headings are present because the first row contains text and not data. For information on copying the peaks list without headings, see Section 3.3.3, Saving the Peak List. To copy the peak list for the displayed peaks to the Windows clipboard: 1. Set peak detection as needed to create a peak list. See Section 3.2, Peak Detection. 2.
Managing Files Copy mass list 2. Select the trace window to copy. 3. Display the peak list by selecting Output Window from the Display menu, then clicking the Chro Peak List or Spec Peak List tab. 4. Sort the peak list as needed. See “Sorting the peak list” on page 3-42. 5. From the Edit menu, select Copy, then select All Peaks. 6. Paste the data into an appropriate application, for example, Microsoft Excel.
Chapter 1 Data Explorer™ Basics 1 1-42 Applied Biosystems
Chapter 2 Using Chromatogram and Spectrum Windows 2 This chapter contains the following sections: 2.1 Opening and Closing Data Files ......................................... 2-2 2.2 Adjusting the Display Range ............................................ 2-11 2.3 Organizing Windows ........................................................ 2-13 2.4 Manipulating Traces ......................................................... 2-14 2.5 Working with Multiple Data Files ...............................
Chapter 2 Using Chromatogram and Spectrum Windows 2.1 Opening and Closing Data Files This section includes: • • • • • • 2 Opening data files Displaying Mariner UV traces Displaying Voyager chromatograms Viewing read-only files Moving between open files Closing data files 2.1.
Opening and Closing Data Files . 2 Figure 2-1 Select Files Dialog Box 2. Click the down arrow to display the Files of type list, then select the file extension to display. 3. Select up to eight data files to open, then click Add or Add All. Add All is not active if the number of selected files exceeds eight. NOTE: You can also select files by double-clicking the file name in the file list. The files are listed in the Files Selected box. Displaying acquisition comment 4.
Chapter 2 Using Chromatogram and Spectrum Windows Specifying settings 5. Select a Restoring Graphics and Processing Settings option to apply to new files you are opening (settings are not applied to files in the list that are already open): • Use Settings from Data File—Applies the last settings used on the data. • Use Default Settings—Applies settings from the appropriate default .SET file for your system. See “Default processing and graphic settings” on page 1-18.
Opening and Closing Data Files 2 Figure 2-2 Data Explorer Window with Four Mariner Data Files Open (Each .
Chapter 2 Using Chromatogram and Spectrum Windows The data is labeled accordingly: Type of Data Mariner data Spectrum Trace Labels If you open a data file you previously calibrated in Data Explorer, all spectra in the data file are calibrated and displayed with an MC or AC trace label. If AutoSaturation Correction is turned on, all spectra in the data file are corrected and displayed with an ASC trace label. For more information, see Section 5.11, AutoSaturation Correction (Mariner Data Only).
Opening and Closing Data Files 2.1.3 Displaying Voyager Chromatograms To display chromatograms for multispectrum Voyager .DAT files: 1. Open the .DAT files as described in “Opening Data Files” on page 2-2. Spectrum traces for the .DAT files are displayed. 2. To display a Chromatogram window, click a Spectrum window to activate it, then select Restore Chromatogram from the View menu. 2 A Chromatogram window is displayed for the data file.
Chapter 2 Using Chromatogram and Spectrum Windows 2.1.5 Moving Between Open Files You can have more than one file open at a time. You can move between open files in three ways: • • • Using tabs 2 Select the open file to view from the Window menu Use tabs Use the Activate File dialog box To move between open files using tabs, click the tab at the bottom of the Data Explorer window (Figure 2-4) to activate the file.
Opening and Closing Data Files 2 Figure 2-5 Select File to Activate Dialog Box 3.
Chapter 2 Using Chromatogram and Spectrum Windows 2.1.6 Closing Data Files You can close files in the following ways: • Select Close from the File menu to close the active file. • Select Close All Files from the File menu to close all files. • In the Open File dialog box, select any open files from the Files Selected list, click Remove, then click Finish to close the selected files. • Select Exit from the File menu to close all files and exit the software.
Adjusting the Display Range 2.2 Adjusting the Display Range To set the display range: 1. Click the Chromatogram or Spectrum window to activate it. 2. From the Display menu, select Range. 3. Select X Range to set the x-axis range. The scaling units depend on the window you are scaling: • Chromatogram—Scales in the same units currently displayed in the Chromatogram window (Spectrum Number or Time [Mariner data only]) • Spectrum—Scales in m/z units 4. Select Y Range to scale the y-axis range.
Chapter 2 Using Chromatogram and Spectrum Windows 5. Set the parameters described below as needed: Parameter Description Scaling Mode 2 Display Relative Autoscales the trace to the largest peak in the selected range. Base Peak Relative Autoscales the trace to the base peak in the entire range, not the selected range. Displays a right-axis label with the base peak intensity.
Organizing Windows 2.3 Organizing Windows Linking views Linking Chromatogram or Spectrum windows in different data files allows you to zoom on multiple data files. NOTE: When different data files are linked, zooming functions performed on one data file are applied to all linked files. Processing and peak centering functions are applied to the active file only. To link data files: 1. Open the data files you want to link. 2.
Chapter 2 Using Chromatogram and Spectrum Windows 2.4 Manipulating Traces This section includes: • • • • • • • • • • • 2 Zooming centering, and customizing a trace Duplicating a trace Dividing the active trace Adding traces from the same data file to a window Removing traces Expanding and linking traces Recalling and rearranging traces (Processing History) Overlaying traces Annotating traces Viewing trace labels Printing traces 2.4.
Manipulating Traces Centering a peak in the trace To center a peak in the trace window: 1. Display the trace containing the peak of interest. 2. Click the Spec Peak List or Chro Peak List tab in the Output window. 3. Double-click the peak to center. The peak is centered in the active trace. Customizing a trace See Section 1.5, Setting Graphic Options, for information on customizing trace parameters such as line widths and data compression. 2.4.
Chapter 2 Using Chromatogram and Spectrum Windows For example, if you select Divide Active Trace to Four when the active trace has a range of 0.0 to 20.0, the active trace is divided into four traces: • • • • 2 First trace represents the range from 0 to 5 Second trace represents the range from 4 to 10 Third trace represents the range from 9 to 16 Fourth trace represents the range from 15 to 20 NOTE: To restore the display to a single trace, select Remove Inactive Traces from the Display menu. 2.4.
Manipulating Traces When you perform certain functions (for example, smoothing), a new trace is created. You can set the Replace mode to add to or replace the active trace. You can add up to seven new traces to a window to allow you to keep original data displayed when you generate new traces. Setting the Replace mode To set the Replace mode: 1. From the Display menu, select Add/Remove Traces. The Display Trace dialog box is displayed (Figure 2-7).
Chapter 2 Using Chromatogram and Spectrum Windows 2. Select the Replace Mode: • Replace the Active Trace (default)—Replaces the active trace with the newly created trace. • Add a New Trace—Adds the newly created trace to the window. The original trace remains displayed and accessible, allowing visual comparison of the traces. 2 NOTE: If Replace Mode is set to Add a New Trace and eight traces are present when you perform a function, the active trace is replaced when a new trace is generated.
Manipulating Traces Original trace 2 Added traces Figure 2-8 Adding Traces (Four Traces Shown, up to Four More Can Be Added) When you perform a function that adds a new trace, the label of the trace changes from Not Used to the label for the type of trace created (Figure 2-9).
Chapter 2 Using Chromatogram and Spectrum Windows Figure 2-9 shows the original trace and three added traces that now contain a smoothed spectrum (SM), a centroided spectrum (CT), and a baseline offset spectrum (BO). Original trace 2 Added traces Figure 2-9 Added Traces Containing Data For a description of trace labels, see “Viewing Trace Labels” on page 2-30.
Manipulating Traces 2.4.5 Removing Traces Removing the active trace To remove the active trace from a window: 1. Click the trace to remove. 2. Click in the toolbar, or right-click the trace, then select Remove Trace from the menu. The trace is removed. Removing inactive traces 2 To quickly remove all inactive traces from the window: 1. Click the trace to keep displayed to make it the active trace. 2. Select Remove Inactive Traces from the Display menu. 2.4.
Chapter 2 Using Chromatogram and Spectrum Windows 2.4.7 Recalling and Rearranging Traces (Processing History) Overview The Chromatogram and Spectrum windows can display up to 8 traces at a time for a data file. However, up to 16 traces are held in memory. You can recall traces previously displayed for an open data file, or rearrange the order of traces, using the Processing History command. You can also set Processing History Options to automatically delete history or to disable the function completely.
Manipulating Traces Setting Processing History options To set Processing History options: 1. From the Tools menu, select Processing History Options. The Processing History Options dialog box (Figure 2-10) is displayed. 2 Figure 2-10 Processing History Options Dialog Box 2. Turn Processing History on or off. If you turn on, specify: • Purge processing history—Records all processing functions performed. Stores the history log in a .CTS file and purges the history log when you close the data file.
Chapter 2 Using Chromatogram and Spectrum Windows 2.4.8 Overlaying Traces This section includes: • • • • • 2 Overlaying traces from different data files Overlaying traces from different data files Overlaying traces in a single data file Changing the active trace Sequentially activating overlaid spectra Setting overlay attributes To overlay traces from different data files: 1. Copy chromatogram or spectrum traces you want to overlay into a trace window. For more information, see Section 2.5.
Manipulating Traces NOTE: When saving results, only the results for the active trace are saved. Overlaying traces in a single data file To overlay traces in a single data file: 1. Display the chromatogram or spectrum traces you want to overlay. For more information, see Section 2.4.4, Adding Traces from the Same Data File to a Window 2. To use settings other than defaults, set attributes for the overlay if needed. See “Setting overlay attributes” on page 2-26. 3.
Chapter 2 Using Chromatogram and Spectrum Windows Changing the active trace 2 Sequentially activating overlaid spectra To change the active trace in an overlay: 1. From the Display menu, deselect Overlay. 2. Click the trace to activate. 3. From the Display menu, select Overlay. When you display overlaid traces, you can sequentially activate each trace by clicking and . NOTE: If you overlay processed spectra, and then display a different spectrum number, the processed spectrum is lost.
Manipulating Traces NOTE: You must select the Use same settings for all traces check box before selecting options for traces. If you do not, settings are applied only to the active trace. 5. In View Setup, select Overlay Trace scaling: • Display Relative—Autoscales each trace to the base peak in the display range • BP Relative—Autoscales each trace to the base peak in the trace • Absolute Value—Maintains scaling of individual traces 6.
Chapter 2 Using Chromatogram and Spectrum Windows 2.4.9 Annotating Traces You can add text annotation to traces by: 2 Copying results • Copying a line of results from the Output window (or copying any ASCII text from any source), then pasting the copied information on the trace • Typing text on the trace To copy results: 1. Generate results as desired by selecting commands from the Tools or Applications menu. For more information, see: • Section 5.3, Manual Calibration, or Section 5.
Manipulating Traces Annotating the trace To annotate the trace: 1. Click the trace at the location where you want to insert text. 2. Right-click, then select: • Paste text—If you copied results. • Add text annotation—If you want to type in text. Type in text as needed. 2 The text is added to the trace, and remains in the Spectrum window until you delete it. NOTE: The text is associated with the x-coordinate. If you display another spectrum, the text remains in view.
Chapter 2 Using Chromatogram and Spectrum Windows 2.4.10 Viewing Trace Labels The Data Explorer software includes a label in the trace header to identify the type of data displayed. NOTE: Trace labels are applied by the software and cannot be removed.
Manipulating Traces Chromatogram Trace Label Description + XAC Realigned extracted absorbance chromatogram XIC xxx ± yyy Extracted ion chromatogram for a selected mass where xxx is the center mass and yyy is the specified window NOTE: Extracted ion chromatograms were previously labeled with “Mass” instead of “XIC”. Figure 2-11 illustrates an extracted ion chromatogram with a “XIC” chromatogram trace label.
Chapter 2 Using Chromatogram and Spectrum Windows Spectrum Trace Label 2 Type of Processing BPI Base peak intensity CT Centroid DAD Diode array data DECONV (Mariner data only) Zero charge deconvoluted trace DI Deisotoped trace ISO Isotope MC Manually mass calibrated NF Noise-filtered trace NRX Noise-removed trace, where X is the number of standard deviations of noise removed RSMX (does not apply to Voyager PSD data) Default smoothing applied, where X is the resolution value (from pea
Manipulating Traces Figure 2-12 illustrates a smoothed spectrum with an “SM5” spectrum trace label. Trace label 2 Figure 2-12 Spectrum Trace Label 2.4.11 Printing Traces Printing traces To print traces: 1. Display the traces to print. To obtain a clear printout, you can set the Trace Color and other attributes to dark colors before printing traces by selecting Default from the Display menu, then selecting White Background.
Chapter 2 Using Chromatogram and Spectrum Windows 3. To print with the x-axis along the longest length of the paper, select Print Setup from the File menu, then select Landscape orientation. NOTE: The Landscape printing orientation you set in Data Explorer is lost when you close Data Explorer. To permanently set the printer, see “Dedicating a printer to landscape orientation” on page 2-35. 2 4.
Manipulating Traces NOTE: Line Widths of 0 or 1 (or lines set to the color white) may not print on certain printers. If traces do not print, change the line width (or color). Dedicating a printer to landscape orientation To dedicate the printer to landscape orientation: 1. From the Windows desktop, click Start, then select Settings. 2. Click Printers. 3. Select the printer name in the displayed list. 4. Click File, then select Document Defaults. 5.
Chapter 2 Using Chromatogram and Spectrum Windows 2.5 Working with Multiple Data Files When you have multiple data files open, you can: 2 • Work with the data files separately to view, zoom, and print • Copy traces from one data file to another to compare or combine data 2.5.1 Working with Separate Data Files To view, zoom, and print multiple data files: 1. Open the data files as described in Section 2.1, Opening and Closing Data Files. 2.
Working with Multiple Data Files NOTE: If you select Print All Views when more than two data files are open, certain printers may not print the data file name. To ensure data file names are printed, print views individually, or only open two data files before you select Print All Views. 2 2.5.2 Copying Traces from Multiple Data Files to a Window You can copy up to seven traces from open data files to a different trace window: 1. Select the trace to copy. 2.
Chapter 2 Using Chromatogram and Spectrum Windows Comparing copied traces After you copy a trace to another trace window, you can compare traces by overlaying (see Section 2.4.8, Overlaying Traces) or by using trace arithmetic (see Section 5.12, Adding and Subtracting Raw or Processed Spectra from the Same or Different Data Files (Dual Spectral Trace Arithmetic). 2.6 Saving, Opening, and Deleting .DAT Results 2 Saving results for .DAT files To save results for .DAT files: 1.
Exporting, Opening, and Deleting .RCD and .RSD Results Files (Mariner Data Only) 2.7 Exporting, Opening, and Deleting .RCD and .RSD Results Files (Mariner Data Only) Exporting results for .RCD and .RSD files To export results to a .RSD or .RCD file: 1. With a data file open in Data Explorer, select a spectrum or chromatogram. 2. From the File menu, select Export, then Result Chromatogram or Result Spectrum. 2 A Save As dialog box appears. Opening results for .RCD and .RSD files 3.
Chapter 2 Using Chromatogram and Spectrum Windows Deleting results for .RCD and .RSD files Use Windows NT Explorer to delete .RCD and .RSD result files. 2.8 Saving, Opening, and Deleting .SPC Results Files (Mariner Data Only) 2 Saving results for .SPC files To save results for .SPC files (Mariner data only): 1. Process a data file to generate results as needed. 2. From the File menu, select Result Spectrum or Result Chromatogram, then select Save As. 3.
Saving, Opening, and Deleting .SPC Results Files (Mariner Data Only) 3. Select the .RST or .RCT file to open, then click OK. NOTE: Saturation Correction is not applied to Mariner .RST files. For more information, see Section 5.11, AutoSaturation Correction (Mariner Data Only). 4.
Chapter 2 Using Chromatogram and Spectrum Windows 2 2-42 Applied Biosystems
Chapter 3 Peak Detection and Labeling 3 This chapter contains the following sections: 3.1 Overview ......................................................... 3-2 3.2 Peak Detection ................................................ 3-6 3.3 Peak List ....................................................... 3-37 3.4 Deisotoping a Spectrum................................. 3-45 3.5 Peak Labeling ................................................ 3-52 3.
Chapter 3 Peak Detection and Labeling 3.1 Overview This section includes: • Default peak detection • The resolution-based peak detection routine 3.1.1 Default Peak Detection Overview When you open a data file, it is automatically peak detected. For: • Chromatographic data—The software uses default settings that have been optimized to yield acceptable peak detection for many sample types. For more information, see Section 3.7, Default Peak Detection Settings.
Overview 3.1.2 The Resolution-Based Peak Detection Routine This section describes: • • • • Type of data affected Process that occurs Detection ranges PSD peak detection for Voyager data Type of data affected The resolution-based peak detection routine applies to spectral data only. You can enable or disable the resolution-based peak detection routine as described on page 3-14.
Chapter 3 Peak Detection and Labeling The software uses the following formula to calculate the expected number of data points in a peak: Expected number of data points Flight time to which the data point corresponds = (2 × mass resolution × Bin size) Where: Mass resolution is a user-defined value. Data-type-dependent defaults are provided, but can be overwritten. Bin size is an instrumental constant: · For Mariner data, Bin size = 1 ns.
Overview Overlapping peak detection ranges To accommodate spectral peaks that occur on the boundary of two peak detection ranges, the software creates detection ranges that overlap (Figure 3-2).
Chapter 3 Peak Detection and Labeling 3.2 Peak Detection This section includes: • • • • • Strategy for Mariner peak detection Strategy for Voyager peak detection Setting peak detection parameters Peak detection parameter descriptions Charge state determination and examples 3.2.1 Strategy for Mariner Peak Detection This section gives some quick suggestions on how to approach Mariner peak detection. For details on peak detection, see Section 3.2.3, Setting Peak Detection Parameters.
Peak Detection 3. If peak detection is not acceptable, leave the Use Resolution Dependent Settings option enabled, and adjust the following global threshold parameters in the following order: • %Base Peak Intensity—Use this parameter to eliminate peaks with an intensity below the specified threshold. This threshold is represented as a percentage of the intensity of the base peak. • %Max Peak Area—Use this parameter to fine-tune noise discrimination on the baseline or noise spikes on peaks.
Chapter 3 Peak Detection and Labeling 3.2.2 Strategy for Voyager Peak Detection This section gives some quick suggestions on how to approach Voyager peak detection. For details on peak detection, see Section 3.2.3, Setting Peak Detection Parameters. Default peak detection values are listed in Section 3.7, Default Peak Detection Settings. Strategy When detecting peaks in Voyager data: 1. Open the data file and observe the effects of the default resolution-dependent settings.
Peak Detection • Deisotope (reflector data only)—Peak deisotoping reduces the spectrum to a monoisotopic centroided plot of the monoisotopic masses. This is useful in identifying overlapping isotope clusters. Make sure peak detection thresholds are set low enough to detect the monoisotopic peak before deisotoping. For more information, see Section 3.4, Deisotoping a Spectrum. Deisotoping provides no benefit on linear data (non-isotopically resolved) or on PSD data (pure isotope data). 3.
Chapter 3 Peak Detection and Labeling Problem Suggested Actions Partially resolved peaks not detected If peaks represent two compounds, and you want both peaks labeled, do either of the following: • Set %Max Peak Area to 0, then adjust the %Base Peak Intensity until peaks are detected. • Click the Peak Processing tab, then change the default Integration Baseline Setting from Valley-to-Valley to Valley-to-Baseline.
Peak Detection 3.2.3 Setting Peak Detection Parameters This section includes: • Before you begin • Setting chromatogram parameters • Setting Basic Settings (spectrum data) • Setting Peak Processing parameters (spectrum data only) • Setting Advanced Settings (spectrum data only) • Resetting Basic Settings Before you begin Before setting peak detection, read: • Section 3.2.1, Strategy for Mariner Peak Detection • Section 3.2.
Chapter 3 Peak Detection and Labeling Figure 3-3 Chromatogram Peak Detection Setup Dialog Box 3 4. Select a detection range and set parameters as needed. 5. To apply settings to all traces, select Use same settings for all traces in view. To set parameters independently for all traces in a window, deselect Use same settings for all traces in view. 6.
Peak Detection Setting Basic Settings (spectrum data) Basic Settings should provide acceptable peak detection for most applications. After you apply these parameters, no further adjustment should be required. If further adjustment is required, select Use Advanced Settings and adjust parameters as needed. To set Basic Settings parameters for spectrum data: 1. Click the Spectrum window to activate it. 2. Click the trace of interest. 3. Click in the toolbar or select Peak Detection from the Peaks menu.
Chapter 3 Peak Detection and Labeling Figure 3-4 Spectrum Peak Detection Setup— Basic Settings Tab 3 5. If you are detecting PSD data, or want to override the Global Thresholds, select Use Advanced Settings and skip to step 7. 6.
Peak Detection NOTE: Resolution-dependent settings do not apply to Mariner chromatogram data or Voyager PSD data. For more information, see Section 3.1.2, The Resolution-Based Peak Detection Routine. 7. To apply settings to all traces, select Use same settings for all traces in view. To set parameters independently for all traces in a window, deselect Use same settings for all traces in view. 8. Type the Mass Resolution value to use for peak detection.
Chapter 3 Peak Detection and Labeling Setting Peak Processing parameters (spectrum data only) To set Peak Processing parameters: 1. Click the Peak Processing tab in the Spectrum Peak Detection Setup dialog box. The Peak Processing tab is displayed (Figure 3-5). 3 Figure 3-5 Spectrum Peak Detection Setup— Peak Processing Tab NOTE: You can enable the %BP Intensity Threshold Cursor and click-drag it to adjust the %Base Peak Intensity. 2.
Peak Detection Setting Advanced Settings (spectrum data only) To set Advanced Settings that you can apply locally to a selected detection range, and that override the thresholds set in the Basic Settings tab: 1. In the Basic Settings tab, select Use Advanced Settings. 2. Click the Advanced Settings tab in the Spectrum Peak Detection Setup dialog box. NOTE: If you select Use Resolution Dependent Settings in the Basic Settings tab, Basic Settings override Advanced Settings.
Chapter 3 Peak Detection and Labeling 3. Select a detection range, then set parameters as needed. 4. Click Apply to accept the parameters and leave the dialog box open, or click OK to accept the parameters and close the dialog box. For a description of the parameters and the data cursor, see “Advanced Settings (spectrum data only)” on page 3-28.
Peak Detection 3.2.4 Peak Detection Parameter Descriptions This section describes: • • • • Chromatogram settings Chromatogram settings Basic Settings (spectrum data) Peak Processing parameters (spectrum data only) Advanced Settings (spectrum data only) Table 3-1 describes the parameters in the Chromatogram Peak Detection Setup dialog box (see Figure 3-3 on page 3-12). Default peak detection values are listed in Section 3.7, Default Peak Detection Settings.
Chapter 3 Peak Detection and Labeling Table 3-1 Chromatogram Settings (Continued) Parameter Description Detection Ranges To delete a range, select the range, then click . (continued) To combine all ranges in the list into one range, click . The peak detection settings displayed in the dialog box correspond to the selected range. To view peak detection settings for another range, select the range of interest.
Peak Detection Table 3-1 Chromatogram Settings (Continued) Parameter Filter Width (continued) Description Hint: Set Filter Width to a number equal to the number of points across the peak. To determine the number of points across a peak, you can change the trace display from Line to Vertical Bars. Each vertical bar represents one data point. For more information, see Section 1.4, Customizing the Data Explorer Window.
Chapter 3 Peak Detection and Labeling Basic Settings (spectrum data only) Table 3-2 describes the parameters in the Basic Settings tab of the Spectrum Peak Detection Setup dialog box (see Figure 3-4 on page 3-14). Default peak detection values are listed in Section 3.7, Default Peak Detection Settings. Table 3-2 Basic Settings Tab Parameters (Spectrum Data Only) Parameter Description Global Thresholds 3 %Base Peak Intensity Specifies a percentage of the base peak intensity as the threshold value.
Peak Detection Table 3-2 Basic Settings Tab Parameters (Spectrum Data Only) (Continued) Parameter %Max Peak Area Description Specifies a percentage of the peak with the largest area as the threshold value. To be detected, peaks must be above this threshold and above the %Base Peak Intensity value. %Max Peak Area is calculated above the local baseline and can compensate for problems related to a rising global baseline.
Chapter 3 Peak Detection and Labeling Table 3-2 Basic Settings Tab Parameters (Spectrum Data Only) (Continued) Parameter Description Peak Resolution Mass Resolution Value used to determine the Filter Width used for detection. For more information, see Section 3.1.2, The ResolutionBased Peak Detection Routine. Default values for different types of data are: 3 • Mariner data—5,000, which is optimized for masses below 3,000 m/z. Decrease this value if you are analyzing proteins.
Peak Detection Table 3-2 Basic Settings Tab Parameters (Spectrum Data Only) (Continued) Parameter Description Trace Settings Use same settings for all traces in view Applies settings to all traces in the active window. NOTE: In previous versions of Data Explorer software, peak detection allowed you to specify Peak Width. The software now automatically uses a minimum peak width that is equal to the Filter Width and a maximum peak width of 10,000 data points.
Chapter 3 Peak Detection and Labeling Peak Processing parameters (spectrum data only) Table 3-3 describes the parameters in the Peak Processing tab of the Spectrum Peak Detection Setup dialog box (see Figure 3-5 on page 3-16). Default peak detection values are listed in Section 3.7, Default Peak Detection Settings.
Peak Detection Table 3-3 Peak Processing Parameters (Spectrum Data Only) (Continued) Parameter Description Charge State Determination (Spectrum only) Maximum Charge State Determines the peak spacing evaluated for the presence of isotope peaks. The expected peak spacing is determined by the Max Charge State plus or minus a tolerance value. The tolerance is calculated as (proton mass/charge state) × 15%. For example, with a Max Charge State of 3, the software checks for peaks that are 0.
Chapter 3 Peak Detection and Labeling Advanced Settings (spectrum data only) Table 3-4 describes the parameters in the Advanced Settings tab of the Spectrum Peak Detection Setup dialog box (see Figure 3-6 on page 3-17). Default peak detection values are listed in Section 3.7, Default Peak Detection Settings. NOTE: If you select Use Resolution Dependent Settings in the Basic Settings tab, Basic Settings override Advanced Settings.
Peak Detection Table 3-4 Advanced Settings (Spectrum Data Only) (Continued) Parameter Detection Ranges (continued) Description You can set multiple, non-contiguous ranges and define parameters for each range independently. You select a range in the Detection Ranges list box by single-clicking the range number. To add a detection range, do one of the following: • Select an existing range, then click .
Chapter 3 Peak Detection and Labeling Table 3-4 Advanced Settings (Spectrum Data Only) (Continued) Parameter Description Active Range Thresholds NOTE: These settings apply to the Detection Range selected, and override the Global Thresholds specified on the Basic Settings tab (described on page 3-19). %BP Intensity See “%Base Peak Intensity” on page 3-20. %Max Peak Area See “%Max Peak Area” on page 3-20. Minimum Intensity Specifies the absolute peak intensity below which peaks are not detected.
Peak Detection Table 3-4 Advanced Settings (Spectrum Data Only) (Continued) Parameter Description Filter Settings Width Number of data points used in smoothing for peak detection before integration. This value is automatically calculated by the software if you select Use Resolution Dependent Settings on the Basic Settings tab. For more information, see Section 3.1.2, The Resolution-Based Peak Detection Routine. NOTE: If you set Filter Width too high, narrow peaks may not be detected.
Chapter 3 Peak Detection and Labeling 3.2.5 Charge State Determination and Examples NOTE: Isotope-resolved peaks in Voyager data are typically singly charged. See Section 3.7, Default Peak Detection Settings, for recommended settings. This section includes: • Charge state determination • Charge state parameter examples • Charge state determination troubleshooting Isotopes Charge state determination 3 For information on isotopes, see Appendix B, Overview of Isotopes.
Peak Detection Max Charge State and Max Isotope # set correctly When the Max Charge State and Max Isotope # are set correctly (in the example shown in Figure 3-7, both are set to 4), the neurotensin 558 m/z isotope cluster contains four peaks at charge state 3. Peaks are 0.33 m/z apart (Figure 3-7).
Chapter 3 Peak Detection and Labeling . Figure 3-8 Max Charge State Set Too Low and Max Isotope # Set Correctly The grouping of the first and fourth peaks into an isotope cluster is apparent when you turn on monoisotopic peak list filtering (Figure 3-9). The software labels the first peak, but removes the mass and charge state labels from the 559.3 m/z peak, indicating that it is part of the 558.3 m/z isotope cluster.
Peak Detection Max Isotope # set too low If the Max Isotope # is set too low, the software incorrectly groups peaks into different isotope clusters. In this example with the Max Isotope # set to 2 and Max Charge State set to 4, the software labels all peaks as charge state 3 (Figure 3-10). Figure 3-10 Max Isotope # Set Too Low, Max Charge State Set Correctly The effect of setting Max Isotope # too low is apparent when you turn on Monoisotopic peak list filtering.
Chapter 3 Peak Detection and Labeling Effect of Minimum Intensity The Minimum Intensity setting on the Advanced Settings tab can also determine how charge states are determined for a peak, because it determines if the software can find a match for a peak. Figure 3-12 illustrates two peaks correctly labeled with charge state 1.
Peak List 3.3 Peak List This section describes: • • • • Displaying the peak list Inserting peaks in the peak list Saving the peak list Sorting, filtering, and printing the peak list 3.3.1 Displaying the Peak List After peak detection, centroiding, and integration, the software creates a peak list for the chromatogram (Mariner data only) and each spectrum in the data file.
Chapter 3 Peak Detection and Labeling Contents of peak list Chromatogram peak list Spectrum peak list 3 3-38 Applied Biosystems The peak lists contain the following information.
Peak List 3.3.2 Inserting Peaks in the Peak List Description Procedure If chromatogram or spectrum peaks are not detected and labeled by the selected detection parameters, you can manually detect and label peaks by inserting peaks in the peak list. To insert peaks: 1. Display the chromatogram or spectrum trace of interest. 2. From the Peaks menu, select Insert Peaks. The Insert Peaks dialog box is displayed (Figure 3-15). 3 Figure 3-15 Insert Peaks Dialog Box 3.
Chapter 3 Peak Detection and Labeling Inserted peaks are: • Removed from the list when you close the data file, reprocess the data, or set peak detection parameters so that the inserted peak is no longer detected. • Assigned a charge state of 0 to indicate the charge state is unknown. CAUTION A zero value in the Spec Peak list does not indicate a charge state of zero. It indicates that the software could not determine the charge state. 3.3.3 Saving the Peak List 3 Saving as a stand-alone .
Peak List NOTE: Inserted peaks are included when you save a .PKT file. NOTE: Peak list headings are not included when you save a .PKT file. If you require headings, copy the peak list directly to Excel instead of saving it as a .PKT file and then importing it. Right-click the peak list to copy. Importing and saving in Excel You can import peak list values into Microsoft Excel to create a spreadsheet. To import and save the peak list in Microsoft Excel: 1. In Excel, select Open from the File menu.
Chapter 3 Peak Detection and Labeling 3.3.4 Sorting, Filtering, and Printing the Peak List Sorting the peak list The peak list is displayed in order by index number. You can sort the list by any field by clicking the column header buttons (Figure 3-16). Click column header buttons to sort by different fields 3 Figure 3-16 Sorting the Peak List Filtering the spectrum peak list Peak list filtering allows you to display only desired peaks in the spectrum peak list.
Peak List 2. Select Enable Peak List Filter, then select: Filter Type Description Monoisotopic Labels the peak of the lowest detected mass in an isotope envelope. Before applying filtering, set the values in Peak Processing detection parameters to accurately detect and label all peaks in the isotope envelope with the correct charge state. If the parameters are not correctly set to yield correct charge states for all peaks, the monoisotopic peak may not be correctly labeled. See Section 3.2.
Chapter 3 Peak Detection and Labeling Printing the peak list Deleting items from the peak list To print the peak list: 1. Display the peak list as you want it printed. 2. Right-click the peak list, then select Print. To delete an item from the peak list, select the item, right-click the spectrum peak list, then select Delete Peak. The entry is removed from the list, and the peak label is removed from the trace.
Deisotoping a Spectrum 3.4 Deisotoping a Spectrum This section includes: • • • • • • • • Description Description During peak deisotoping When to use Requirements Using the Deisotope function Troubleshooting Example Returning to the original spectrum The Deisotope function reduces a spectrum to a centroided plot by deconvoluting the monoisotopic peaks from the current peak list.
Chapter 3 Peak Detection and Labeling If the expected higher theoretical peak masses and areas are present in the peak list: • The peak in question is considered to be a monoisotopic peak. • The intensities of the higher mass peaks that correspond to the expected isotope ratios are combined with the intensity of the peak in question (additional intensity that may be related to a contaminant or an overlapping isotope envelope is not combined, and will be evaluated in the next iteration).
Deisotoping a Spectrum Peaks in original trace Deisotoped trace Increased amplitude of first peak indicates it is a monoisotopic peak If first three peaks are part of the same isotope cluster Contribution to expected isotope ratio by contaminant Peak that is not part of cluster Increased amplitude of first peak indicates it is a monoisotopic peak If first three peaks are part of the same isotope cluster, but contaminant is also present Contaminant Peak that is not part of cluster Increased amplitud
Chapter 3 Peak Detection and Labeling Using the Deisotope function To use the Deisotope function: 1. Display the spectrum trace of interest. 2. Make sure peak detection thresholds are set low enough to detect the monoisotopic peak before deisotoping. If the detection thresholds are not set low enough, adjust them. For information, see Section 3.2.3, Setting Peak Detection Parameters.
Deisotoping a Spectrum CAUTION If you enter an invalid value in the Adduct field, for example, numbers, the spectrum is still converted to a deisotoped spectrum, and the peak height is proportional to the original peak area. 7. In the Formula text box, type the generic formula (using any elements in the Periodic table) that approximates the isotopic pattern for the compound class you are examining. Generic formulas include: • • • • 8.
Chapter 3 Peak Detection and Labeling Example Figure 3-19 and Figure 3-20 illustrate the effects of deisotoping. Before deisotoping (Figure 3-19), the spectrum includes an isotope pattern with four detected peaks. Figure 3-19 Spectrum Before Deisotoping After deisotoping (Figure 3-20), the trace includes two labeled centroid bars that represent monoisotopic masses, indicating that the original trace represents two isotopic envelopes.
Deisotoping a Spectrum Returning to the original spectrum To return to the original spectrum: • If the original spectrum was an unprocessed spectrum, select Spectrum Number from the Display menu. The number of the original spectrum is displayed in the Select Spectrum dialog box. Click OK. • If the original trace was a processed spectrum, select Processing History from the Display menu, then select the original trace.
Chapter 3 Peak Detection and Labeling 3.5 Peak Labeling This section includes: • Charge state labels • Setting chromatogram and spectrum peak labels • Setting custom peak labels Factors affecting peak labeling Peak labels are displayed only for detected peaks in the peak list (displayed in the Output window). Thresholds for peak labeling are set independent of peak detection. Peaks listed in the peak list are determined by: • Peak detection parameters, described in Section 3.
Peak Labeling 3.5.1 Charge State Labels Charge state labels A unique feature of the Data Explorer software is the ability to label multiply charged isotope peaks with their charge state. In general, the observed spacing between isotopes is determined by the charge state of the detected ion. Spacing between isotope peaks is narrower at higher charge states. For more information on isotopes, see Appendix B, Overview of Isotopes.
Chapter 3 Peak Detection and Labeling • Isotopes must be resolved • Peak detection parameters must be set to detect all of the peaks in the isotope cluster • Charge determination parameters must be set appropriately • Charge state labels must be enabled 3.5.
Peak Labeling Figure 3-21 Chromatogram Peak Label Dialog Box 3. Select Enable Labeling. 4. Set the number of decimal points to be displayed. 5. Select Label Attributes: • Overlapping—Allows labels to be displayed when peaks are close together. • Peak bounds—Displays peak start, peak end, and baseline. • Orientation—Specifies Horizontal, 45-degree, or Vertical labels. 6. Select the label content to display, (for example, Spectrum #, Time, Vial Number). 7.
Chapter 3 Peak Detection and Labeling 8. Click OK. The trace is displayed. The detected peaks that meet the peak labeling criteria are labeled. Setting spectrum labels To label spectrum peaks: 1. Click the Spectrum window to activate it. 2. From the Peaks menu, select Peak Label. The Spectrum Peak Label dialog box is displayed (Figure 3-22). 3 Figure 3-22 Spectrum Peak Label Dialog Box 3-56 Applied Biosystems 3. Select Enable Labeling to label peaks in m/z format. 4.
Peak Labeling Label Mass 5. Select the Mass Type (peak apex or peak centroid). 6. Select the Peak Mass Label Type: Description Labels with Apex or Centroid mass. NOTE: If you create a custom user label for a mass, the user label is displayed instead of the mass. For more information, see Section 3.5.3, Setting Custom Peak Labels. Mass difference from the selected peak NOTE: This label was previously called Mass Offset.
Chapter 3 Peak Detection and Labeling 7. Select label attributes: • Overlapping—Allows labels to be displayed when peaks are close together. • Peak bounds—Displays peak start, peak end, and baseline. • Orientation—Specifies Horizontal, 45-degree, or Vertical labels. 8. Select label content to be displayed in addition to m/z: • Area—Integrated area of peak, displayed with an “A” label.
Peak Labeling If peak list filtering is enabled, only the detected peaks that meet the peak filtering criteria are labeled according to the peak label settings. Otherwise, all detected peaks are labeled according to the peak label settings. See Section 3.2.4, Peak Detection Parameter Descriptions, and “Filtering the spectrum peak list” on page 3-42.
Chapter 3 Peak Detection and Labeling Charge state not displayed If no charge is displayed, there are a few possible causes: • Peaks are more than 1 Da apart. • Filter width is set too high to detect other isotope peaks. • The maximum charge state for charge state determination is set lower than the charge state of the peak. • Charge state determination parameters are set such that peaks are determined to have no charge. See Section 3.2.5, Charge State Determination and Examples.
Peak Labeling 3.5.3 Setting Custom Peak Labels This section includes: • • • • • • Description Description Customizing colors, font, and size Creating custom peak labels Applying user labels from .LBC or LBS files Displaying user labels User labels not displayed A custom peak label displays the label name you enter. If you specify a custom label for a spectrum, it is displayed instead of the mass value.
Chapter 3 Peak Detection and Labeling Column header buttons Figure 3-23 User Label Setup Dialog Box 3 4. Select the Label Type (spectra only): • Mass—Labels with Apex or Centroid mass. • Mass difference from adjacent peaks—Applies the labels to peaks that have the specified mass difference relative to the adjacent labeled peak of lower m/z. 5. Select Match Charge State (spectra only) if you want the charge state of a peak evaluated before applying the user label.
Peak Labeling 6. To manually enter label settings, click . The User Label Entry dialog box is displayed (Figure 3-24). Figure 3-24 User Label Entry Dialog Box (Spectrum) 7. Set the following parameters: 3 Spectrum Parameter Description/Specifies Label Text of the label to display. Peak Mass (if Label Type selected is Mass) Mass of the peak to which the label applies. Mass Difference (if Label Type selected is Difference) Difference in mass that must exist between peaks to apply user labels.
Chapter 3 Peak Detection and Labeling Chromatogram Parameter Description Label Text of the label to display. X Value Retention time or Spectrum Number of the peak to label. The default units for X Tolerance correspond to the units of the x-axis. X Tolerance Retention time or Spectrum Number that the peak must fall within to apply user labels. To enter Peak Mass, X Value, or Mass Difference, you can type values or right-click-drag over a peak.
Peak Labeling Applying user labels from .LBC or .LBS files You can apply labels you previously created and saved in .LBC or .LBS files. Open the .LBC or .LBS file by clicking in the User Label Setup dialog box. Select the file, then click Open. The labels are imported into the .DAT file and displayed in the label list. You can modify the settings, delete or add labels, and save the changes in a new .LBC or .LBS file. NOTE: Changes you make to user labels imported into a .DAT file are not saved in the .
Chapter 3 Peak Detection and Labeling User labels not displayed If a user label is not displayed or is displayed incorrectly, possible causes are: • User labels are not enabled. • The peak does not fall within the specified Tolerance. • The label is too long or peaks are too close together. If there is not enough room for the label to be displayed, the label may be suppressed. Zooming the region of interest expands the trace and may allow the labels to be displayed.
Process that Occurs During Peak Detection, Centroiding, and Integration 3.6 Process that Occurs During Peak Detection, Centroiding, and Integration This section gives an overview of the process that occurs during peak detection, centroiding and integration. Parameters are defined in detail in Section 3.2.4, Peak Detection Parameter Descriptions.
Chapter 3 Peak Detection and Labeling • For spectral data, determines the peak boundaries by one of two means: • If the Noise Threshold is greater than zero, the software scans from the valley regions toward the apex region using the number of data points defined by the Filter Width. If the difference between two consecutive filtered regions is greater than the Noise Threshold, the midpoint of the filter region closest to the apex is used as the peak bound.
Process that Occurs During Peak Detection, Centroiding, and Integration After peak detection After peaks are detected: • Centroid mass is calculated for spectral data, then modified by Gaussian peak fitting, if it is selected. • Chromatographic and spectral peaks are integrated. • Peak lists are generated for chromatograms and spectra.
Chapter 3 Peak Detection and Labeling Integration The Data Explorer software integrates chromatographic and spectral peaks to calculate the peak area using one of two methods to determine the peak baseline (Figure 3-25): • Valley-to-baseline—Drops a vertical line from all valleys to a horizontal baseline. The level of the horizontal baseline is determined using the minimum peak valley point (left or right) for each peak. • Valley-to-valley—Forces a baseline to all valley points.
Default Peak Detection Settings 3.7 Default Peak Detection Settings This section includes: • Default .SET files provided • Additional Voyager .SET files provided Default .SET files provided Default peak detection settings are contained in the following .SET files: • • • • MARINER.SET VOYAGERLINEAR.SET VOYAGERREFLECTOR.SET VOYAGERPSD.SET The following table lists the default settings in .SET files provided for chromatograms. Parameter Chromatogram Settings in All .
Chapter 3 Peak Detection and Labeling The following table lists the default settings in the .SET files provided for spectra. Parameter Voyager Spectrum Mariner Reflector Spectrum Linear PSD VOYAGER MARINER.S VOYAGER VOYAGER REFLECTOR ET LINEAR.SET PSD.SET .SET Basic Settings (spectrum data) 3 %Base Peak Intensity 0.25 0 0 0 %Max Peak Area 0.
Default Peak Detection Settings Parameter Voyager Spectrum Mariner Reflector Spectrum PSD Linear VOYAGER MARINER.S VOYAGER VOYAGER REFLECTOR ET PSD.SET LINEAR.SET .SET Advanced Detection Range Resolution dependent Resolution dependent Resolution dependent Five ranges Filter Width Resolution dependent Resolution dependent Resolution dependent Range dependent Increment Resolution dependent 1 1 1 Noise Threshold 0 0 0 0 %Base Peak Intensity 0.25 0 0 0 %Max Peak Area 0.
Chapter 3 Peak Detection and Labeling 3 3-74 Applied Biosystems
Chapter 4 Examining Chromatogram Data 4 This chapter contains the following sections: 4.1 Overview ......................................................... 4-2 4.2 Creating an Extracted Ion Chromatogram ........ 4-5 4.3 Creating an Extracted XAC Chromatogram (Mariner Data Only) ....................................... 4-13 4.4 Noise Filtering/Smoothing .............................. 4-17 4.5 Adding and Subtracting Spectra .................... 4-20 4.
Chapter 4 Examining Chromatogram Data 4.
Overview You can display extracted chromatograms from Mariner data files by selecting the Process menu with a Chromatogram window displayed, then selecting: Select To display Extracted Ion— Center Window or Range Extracted Ion Chromatogram (XIC), which includes only the signal response from a mass window or range. For more information, see Section 4.2.1, Creating an Extracted Ion Chromatogram (XIC).
Chapter 4 Examining Chromatogram Data You can display the following types of Voyager data by selecting Extracted Ion from the Process menu with a Chromatogram window displayed, then selecting: Select To display Center Window or Range (XIC) Extracted Ion chromatogram, which includes only the response from a mass window or range. For more information, see Section 4.2.1, Creating an Extracted Ion Chromatogram (XIC).
Creating an Extracted Ion Chromatogram 4.2 Creating an Extracted Ion Chromatogram This section includes: • Creating an Extracted Ion Chromatogram (XIC) • Creating a Constant Neutral Loss (CNL) Chromatogram You can create an extracted ion chromatogram: • To improve the signal-to-noise ratio for a mass or mass range of interest • To determine if mass differences in a data file correspond to loss of specific fragments by generating a Neutral Loss Chromatogram 4.2.
Chapter 4 Examining Chromatogram Data 4. In the Extracted Ion Chromatogram dialog box (Figure 4-1 on page 4-7), select one of the following from the Mass Range/Difference Type drop-down list: • Center/Window, then type the mass of interest and the mass window for masses to include. NOTE: When analyzing multiple components with similar masses, set a Window of less than 0.5 to include only the mass of interest. • Range, then type the From and To values for masses to include.
Creating an Extracted Ion Chromatogram Figure 4-1 Extracted Ion Chromatogram Dialog Box 5. 6. Specify the Extraction Mode: • Accumulative—Creates one extracted ion chromatogram for all masses entered and sums intensities • Individual—Creates one extracted ion chromatogram for each mass entered 4 Click OK.
Chapter 4 Examining Chromatogram Data From the Spectrum window To create an extracted ion chromatogram for a mass range from the Spectrum window: 1. Click the Chromatogram window to activate it. 2. Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing. 3. In the Spectrum window, right-click-drag over the mass region of interest in the extracted ion chromatogram.
Creating an Extracted Ion Chromatogram 4.2.2 Creating a Constant Neutral Loss (CNL) Chromatogram This section includes: • • • • • Overview Overview Applications Labeling spectrum peaks with mass difference (optional) Procedure Example To rapidly screen for the presence of mass differences corresponding to loss of specific fragments, you can create a Constant Neutral Loss (CNL) extracted chromatogram.
Chapter 4 Examining Chromatogram Data Labeling spectrum peaks with mass difference (optional) You can label spectrum peaks with mass differences to assist you in determining the mass differences to specify in the CNL extracted chromatogram. To label spectrum peaks with mass differences: 1. Display a spectrum of interest from the data file for which you are creating the CNL extracted chromatogram. 2. Click the Spectrum window to activate it, then select Peak Label from the Peaks menu. 3.
Creating an Extracted Ion Chromatogram 5. Type in the Mass Difference and Tolerance. NOTE: Do not right-click-drag across a peak in the Spectrum window to select the mass difference. Figure 4-3 Extracted Ion Chromatogram Dialog Box with Neutral Loss Selected 6. Specify the Extraction Mode: • Accumulative—Creates one extracted ion chromatogram for all mass differences entered and sums intensities • Individual—Creates one extracted ion chromatogram for each mass difference entered 7. Click OK.
Chapter 4 Examining Chromatogram Data Example Figure 4-4 shows a TIC that contains three flavonoid compound peaks. To determine if the diglycosyl group has fragmented from the parent ion in any of these compounds, you can generate a CNL extracted chromatogram. Figure 4-4 TIC for Flavonoid Mixture Containing Three Peaks Figure 4-5 shows a CNL extracted chromatogram generated for mass difference of 308.146 m/z which corresponds to the diglycosyl group.
Creating an Extracted Absorbance Chromatogram (XAC) (Mariner Data Only) 4.3 Creating an Extracted Absorbance Chromatogram (XAC) (Mariner Data Only) This section describes how to create an Extracted Absorbance chromatogram (XAC) for DAD Mariner data: • From the Chromatogram window • From the Spectrum window From the Chromatogram window To create an extracted absorbance chromatogram (XAC) for a selected wavelength window or range: 1. Click the Chromatogram window to activate it. 2.
Chapter 4 Examining Chromatogram Data Figure 4-6 Extracted Absorbance Chromatogram Dialog Box 6. 4 7. Specify the Extraction Mode: • Accumulative—Creates a single trace combining intensities of all specified wavelengths • Individual—Creates one trace for each specified wavelength Click OK. The extracted absorbance chromatograms are displayed in the Chromatogram window with an XAC trace label.
Creating an Extracted Absorbance Chromatogram (XAC) (Mariner Data Only) From the Spectrum window To create an extracted absorbance chromatogram for a wavelength range from the Spectrum window: 1. Click the Chromatogram window to activate it. 2. From the Display menu, select Traces, then select a DAD TAC or DAD Channel chromatogram. The TAC or Sig Absorbance trace appears in the Chromatogram window. 3.
Chapter 4 Examining Chromatogram Data Figure 4-7 Extracted Absorbance Chromatogram 7. 4 4-16 Applied Biosystems To return to the original trace, see “Returning to the original trace” on page 4-4.
Noise Filtering/Smoothing 4.4 Noise Filtering/Smoothing Description The Noise Filter/Smooth command provides three options for reducing noise in chromatogram traces: • Noise filter • Smooth by the Gaussian method • Noise removal Procedure To noise filter or smooth a chromatogram trace: 1. From the Process menu, select Noise Filter/Smooth. The Noise Filter/Smooth dialog box (Figure 4-8) is displayed.
Chapter 4 Examining Chromatogram Data 2. Type of Data Higher resolution data Select the method to use based upon the type of data you are examining, then enter the associated value displayed for the method you select: Suggested Method Noise Filter (NF) (May affect peak resolution.) Higher molecular weight data 4 Gaussian Smooth (SM) (May affect peak resolution.) 4-18 Applied Biosystems Description Specify a Correlation Factor of 0 to 1.0. Settings from 0.5 to 0.
Noise Filtering/Smoothing Type of Data High-resolution data Suggested Method Noise Removal (NR) (Does not affect peak resolution.) Description Specify the number of standard deviations of noise to remove. The software automatically calculates the average white noise for all frequencies across the spectrum, then removes the specified number of standard deviations of noise.
Chapter 4 Examining Chromatogram Data 4.5 Adding and Subtracting Raw Spectra Within a Data File Use the Add/Subtract Spectra command to manipulate raw spectra within a single data file. NOTE: To manipulate processed spectra, spectra from different data files, or spectra acquired under slightly different instrument calibrations, use Trace Arithmetic. For more information, see Section 5.
Adding and Subtracting Raw Spectra Within a Data File Figure 4-9 Add and Subtract Spectra Dialog Box . 4. Select spectra to add by doing one of the following: • Right-click-drag the area of the trace in the Chromatogram window. The numbers of the selected spectra are added to the list window. A range of spectrum numbers is indicated as X:X. For example, 10:20 indicates spectrum number 10 through spectrum number 20. • Click , type the spectrum number range to add, for example 10:20, then press Enter.
Chapter 4 Examining Chromatogram Data NOTE: Before you can subtract spectra, you must first specify spectra to be added. 6. Select spectra to subtract as described in step 4. 7. Select the Add/Subtract mode: • Average—Spectra in each list are averaged before the addition or subtraction occurs. • Accumulate—Spectra in each list are summed before the addition or subtraction occurs.
Displaying MS Method Data (Mariner Data Only) 4.6 Displaying MS Method Data (Mariner Data Only) Overview In this section If you acquired a data file using an MS Method and assigned event tags, you can display chromatogram traces in Data Explorer filtered by event tag.
Chapter 4 Examining Chromatogram Data Hint: Add mode is useful when you filter the same trace for different event tags. The original trace remains displayed and accessible. Each filtered trace (up to four total traces) is added, allowing for visual comparison. Filtering event tags To display chromatogram traces for selected event tags: 1. Display the data file containing the event tags. 2. Click the Chromatogram window to activate it. 3.
Displaying MS Method Data (Mariner Data Only) NOTE: Only tags present in the data file are available. 6. Select the event tags to display, then click OK. The filtered trace is displayed with an EF trace label (Figure 4-11). Figure 4-11 Event Filtered Trace NOTE: The spectra in an event-filtered trace are numbered contiguously (1,2,3...) regardless of their relation to the overall acquisition.
Chapter 4 Examining Chromatogram Data Hint: Add mode is useful when you are filtering the same trace for different event tags. The original trace remains displayed and accessible. Each filtered trace (up to four total traces) is added, allowing for visual comparison. 7. Evaluating filtered traces To return to the original trace, see “Returning to the original trace” on page 4-4.
Adjusting the Baseline 4.7 Adjusting the Baseline This section includes: • Using Baseline Offset • Using Baseline Correction 4.7.1 Using Baseline Offset Use the Baseline Offset command to offset the y-axis in a chromatogram to improve the appearance of a trace or correct a sloping baseline. To use Baseline Offset: 1. Activate the window on which you want to perform the offset. NOTE: You can select a Chromatogram or Spectrum window.
Chapter 4 Examining Chromatogram Data Figure 4-12 Baseline Offset Dialog Box 4. Right-click-drag the left baseline to offset. The selected value is displayed in the Left Baseline field. 5. Right-click-drag the right baseline to offset. The selected value is displayed in the Right Baseline field. 6. To limit the baseline offset to the area between the two selected points, select Only Apply from L to R Baseline Midpoint.
Adjusting the Baseline 4.7.2 Using Baseline Correction Description When to use The Baseline Correction feature is a function that corrects for a curved baseline, including a DC-offset baseline, by eliminating broad artifacts from the data set. Baseline-correct if you are analyzing data: • With a baseline that is not flat, and you are using the %Base Peak Intensity parameter (intensity-based thresholding) to screen out noise peaks. For best results, apply Baseline Correction, then re-detect peaks.
Chapter 4 Examining Chromatogram Data 4.8 Using UV Trace Offset (Mariner Data Only) To align a UV trace with a chromatogram trace: 1. Display the chromatogram and UV traces of interest. 2. Display the x-axis in retention time for each trace by selecting Traces from the Display menu, selecting X Axis In, then selecting Time. 3. Set the Replace Mode to Add a New Trace. For information see “Setting the Replace mode” on page 2-17. 4. From the Process menu, select Realign UV Trace.
Using UV Trace Offset (Mariner Data Only) 6. Click OK. The UV trace peak is shifted to align with the chromatogram trace peak. NOTE: To restore the original UV trace, open the UV Trace Offset dialog box (see step 4), then click Reset. 7. To return to the original trace, see “Returning to the original trace” on page 4-4.
Chapter 4 Examining Chromatogram Data 4 4-32 Applied Biosystems
Chapter 5 Examining Spectrum Data 5 This chapter contains the following sections: 5.1 Overview ......................................................... 5-2 5.2 Creating a Combined Spectrum ...................... 5-4 5.3 Manual Calibration........................................... 5-5 5.4 Automatic Calibration..................................... 5-26 5.5 Centroiding .................................................... 5-36 5.6 Mass Deconvolution (Mariner Data Only) ....... 5-37 5.
Chapter 5 Examining Spectrum Data 5.1 Overview Types of spectra you can display You can display the following types of spectrum data: • Single spectrum—Double-click any point in the TIC to display the corresponding spectrum. • Combined spectrum—Click-drag across a region of the chromatogram to sum the intensity at each mass for all selected spectra. Combining spectra can improve peak shape, signal intensity, and signal-to-noise ratio.
Overview Returning to the original spectrum Many processing functions generate a new trace. If you have Trace Replace mode set to Replace, the new trace replaces the original trace. For information on Replace mode, see Section 2.4.4, Adding Traces from the Same Data File to a Window. To return to the original spectrum: • If the original spectrum was an unprocessed spectrum, select Spectrum # from the Display menu. The number of the original spectrum is displayed in the Select Spectrum dialog box.
Chapter 5 Examining Spectrum Data 5.2 Creating a Combined Spectrum NOTE: Before creating a combined spectrum for Voyager multispectrum data files, calibrate the data. See Section 5.3, Manual Calibration. To create a combined spectrum: 1. Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing. 2. In the Chromatogram window, right-click-drag across the region of the chromatogram that contains the spectra to combine.
Manual Calibration 5.3 Manual Calibration This section describes: • • • • • • Overview of manual calibration Manually calibrating Creating or modifying a calibration reference file Reverting to instrument calibration Hints for calibrating Mariner data Hints for calibrating Voyager data NOTE: Manual calibration is not supported for Mariner DAD data. 5.3.1 Overview of Manual Calibration Overview During manual calibration: • You specify a calibration reference file (.REF) that contains reference masses.
Chapter 5 Examining Spectrum Data The manual calibration feature provides two modes for peak matching: • Automatic—The software automatically compares reference masses to observed masses, and lists peaks that are within the specified peak matching criteria. • Manual—You manually select an observed mass, then select the reference mass for comparison. Hint: It is often useful to automatically perform a match and fit first, then manually adjust the fit as needed. Calibration references files (.
Manual Calibration 5.3.2 Manually Calibrating This section describes: • • • • • Before calibrating Voyager data Before calibrating Voyager data Manually calibrating a single spectrum Applying new constants to the data file Exporting calibration constants (.
Chapter 5 Examining Spectrum Data Manually calibrating a single spectrum NOTE: Multi-point calibration yields higher mass accuracy than one-point calibration. Selecting calibrant peaks that bracket the mass of interest also yields higher mass accuracy. To manually calibrate a single spectrum: 1. Click the Spectrum window to activate it, then select the spectrum trace of interest. 2. From the Peaks menu, select Peak Label, set the Mass Label Type to Centroid, then click OK.
Manual Calibration Figure 5-2 Manual Mass Calibration Dialog Box 4. Select a calibration reference file. For information on creating a reference file, see Section 5.3.3, Creating or Modifying a Calibration Reference File (.REF). 5. Enter reference matching and calibration criteria: • Minimum Intensity—Peaks must be above this intensity to be considered a match. Select the unit for Minimum Intensity, % Relative Intensity or Absolute Counts.
Chapter 5 Examining Spectrum Data 6. Select the Peak Weighting Factor. If the calibration includes more than two points, you can apply the following weighting factors to fit points to the curve: • None—All peaks weighted equally • Inverse Width—Narrower peaks are weighted more than broader peaks • Height—More intense peaks are weighted more than less intense peaks Manually matching peaks 7. To manually select the reference mass for a peak, right-click-drag over the peak of interest.
Manual Calibration 8. Do any of the following: • Click OK to accept the selected reference mass for matching, then add it to the Peaks Matched list. • Select a different reference mass, then click OK. • Type new reference mass information in the Name, Theoretical m/z, Charge, and Elemental Composition text boxes, select the mass type, then click OK to accept the reference mass for matching. NOTE: You must type a minus sign (–) preceding the charge in the Charge text box for negative charge states.
Chapter 5 Examining Spectrum Data 9. Repeat step 7 and step 8 until all desired peaks are in the matched list. Eliminating data 10. To eliminate unacceptable data points from the calibration, do either of the following: points • Select the data point (mass) in the Peaks matched list, then click Delete Selected Match. • Click Eliminate Fit Outlier.
Manual Calibration Eliminate Fit Outlier removes the mass associated with the largest fit error in the Output window, not the mass associated with the largest initial error in the Peaks Matched list. The 267 Da mass is removed when you click Eliminate Fit Outliers because it generated the largest fit error (below). Figure 5-4 Eliminate Fit Outlier Deletes the Match with the Largest Fit Error from the Output Window To clear the entire list, click Delete Entire List. Plotting 11.
Chapter 5 Examining Spectrum Data Calibration statistics Figure 5-5 Calibration Statistics in Output Window If you calibrate more than once, subsequent calibration statistics are added to the end of the list in the Output window. Older calibration statistics are listed at the top of the list. Automatically matching peaks You can automate peak matching by clicking Match Peaks instead of right-click-dragging individual peaks and selecting the mass.
Manual Calibration NOTE: If you are calibrating Mariner data, see “Ensuring that masses match during calibration” on page 5-24. Applying new constants to the data file If you are calibrating To apply the calibration constants to the data: Click The following occurs Mariner data (including MS Method data) Apply Calibration All spectra in the data file are calibrated and displayed with an MC trace label. The calibration constants are saved with the data file.
Chapter 5 Examining Spectrum Data Exporting calibration constants (.CAL file) The calculated calibration constants can be exported to a .CAL file for use with other data files. You have two options for exporting: • Export the current calibration—Click Export in the Manual Mass Calibration dialog box to calculate and export the current calibration constants from the masses displayed in the Peaks Matched list in the Manual Mass Calibration dialog box. NOTE: The calibration constants saved in the .
Manual Calibration 5. If you are calibrating To save the calibration to the data file, select Mass Calibration from the Process menu, then: Select The following occurs Mariner data (including MS Method data) Apply Calibration All spectra in the data file are calibrated and displayed with an MC trace label. The calibration constants are saved with the data file. Each spectrum in the data file is calibrated when displayed.
Chapter 5 Examining Spectrum Data Definition A calibration reference file (.REF) is a list of masses and corresponding information from which you can select reference masses during calibration. .REF files provided The following default calibration reference files are provided with the software: • • • • • Calibration reference file contents MARINER_POS.REF MARINER_NEG.REF VOYAGER.REF ANGIOTENSIN_FRAGMENTS.REF IMMONIUM_IONS.
Manual Calibration The Edit/Create Reference Peak Information dialog box (Figure 5-6) is displayed. Figure 5-6 Edit/Create Reference Peak Information Dialog Box 2. Type the Name and Theoretical m/z for a reference compound, then select the charge state. Optionally, enter the Elemental Composition for the compound. 3. Specify the mass type (Resolved Isotope Mass or Average Mass).
Chapter 5 Examining Spectrum Data CAUTION The software allows you to add multiple items with the same m/z value to the reference file if any other attribute of the reference compound is different (for example, charge state or name). Each mass in the list is considered during calibration. If the mass list contains duplicate entries, the calibration may return an invalid number of matches. 6.
Manual Calibration 3. To modify an entry, click the entry to select it, modify the entry as needed, then click Update. 4. To delete an entry, click the entry to select it, then click Delete. 5. To add an entry, type the Name and Theoretical m/z for a reference compound, then select the charge state. Optionally, enter the Elemental Composition for the compound. 6. Specify the mass type (Resolved Isotope or Average). 7. Click Insert.
Chapter 5 Examining Spectrum Data 5.3.4 Reverting to Instrument Calibration The Revert to Instrument Calibration function does the following: • Mariner data—Reapplies the original calibration constants used to acquire the data. • Voyager data—Applies default calibration to the data, regardless of whether default or external calibration is used to acquire the data. To revert the calibration: 1. From the Process menu, select Mass Calibration, then select Revert to Instrument Calibration.
Manual Calibration If you are calibrating Voyager data Select The following occurs Apply Calibration The current spectrum is calibrated and displayed with an MC trace label. The calibration constants are saved with the spectrum. Apply to All All spectra in the data file are calibrated using the currently displayed calibration, and are displayed with an MC trace label. The calibration constants are saved with the data file. Each spectrum in the data file is calibrated when displayed.
Chapter 5 Examining Spectrum Data 5.3.5 Hints for Calibrating Mariner Data Ensuring that masses match during calibration Mariner TOF Analyzer parameters affect flight times of ions. If you acquired Mariner data using different TOF Analyzer parameters, and did not calibrate the data in the Instrument Control Panel to compensate for the altered parameters, masses in the data file may be significantly different from the reference masses.
Manual Calibration 5.3.6 Hints for Calibrating Voyager Data Importing a calibration If you import a calibration, you must import a calibration generated from a data file that was acquired on the same instrument using identical settings for the following instrument setting parameters: • Polarity • Instrument mode If you import a calibration that was generated using settings that are different from the current Polarity and Instrument Mode settings, an error message is displayed.
Chapter 5 Examining Spectrum Data 5.4 Automatic Calibration This section includes: • Overview of automatic calibration • Importing and specifying automatic calibration settings • Automatically calibrating (Mariner data only) NOTE: Automatic calibration is not supported for Mariner DAD data. 5.4.
Automatic Calibration • Matches all peaks that meet the specified Reference Matching criteria. If the number of peaks matched is greater than or equal to the specified Minimum Number of Peaks to Match, and the resulting fit errors are less than or equal to the specified Max Outlier Error, calibration is successful. • If any points exceed the specified Max Outlier Error, the software eliminates the outliers, one-by-one, worst to best, until all points are within the specified Max Outlier Error.
Chapter 5 Examining Spectrum Data When to use Use automatic calibration for Mariner data when you: • Have many spectra to calibrate • Know in advance what reference masses to use • Know in advance that the quality of the reference mass signals is acceptable For information on manual calibration, see Section 5.3, Manual Calibration.
Automatic Calibration 5.4.2 Importing and Specifying Automatic Calibration Settings Importing Hint: Importing automatic calibration settings is useful when you calibrate batches of related samples. Automatic calibration settings are saved as part of processing settings in a .DAT file. To use auto calibration settings from another .DAT file: 1. Open the data file containing the desired auto calibration settings files. 2. Save the .
Chapter 5 Examining Spectrum Data 2. From the Peaks menu, select Peak Label, then set the Mass Label Type to Centroid. NOTE: For spectra containing broad peaks that have unresolved adducts or impurities such as proteins, you may obtain better results if you use apex instead of centroid settings. 3. From the Process menu, select Mass Calibration, then select Automatic Calibration. The Automatic Calibration Settings dialog box is displayed (Figure 5-7).
Automatic Calibration 5. To add up to 10 reference masses to the Masses to Match list, do either of the following: • Click Add All to add the first 10 reference masses from the reference file. • Click Add Reference To List to individually select reference masses to add. NOTE: If the current list already contains 10 reference masses, you must delete a mass before you click Add References to List.
Chapter 5 Examining Spectrum Data 6. Specify reference masses to add by doing either of the following: • Click a mass, then click OK. • Type new reference mass information in the Name, Theoretical m/z, Charge, and Elemental Composition fields, then click OK. NOTE: You must type a minus sign (–) preceding the charge in the Charge text box for negative charge states. NOTE: You can save changes to the Calibration Reference File by clicking Save.
Automatic Calibration 10. Select the Peak Weighting Factor. If the calibration includes more than two points, you can apply the following weighting factors to fit points to the curve: • None—All peaks are weighted equally • Inverse Width—Narrower peaks are weighted more than broader peaks • Height—More intense peaks are weighted more than less intense peaks 11.
Chapter 5 Examining Spectrum Data 5.4.3 Automatically Calibrating (Mariner Data Only) This section includes: • • • • Automatically calibrating Automatically calibrating Applying new constants to the data file Calibration results Applying auto calibration settings to other files To automatically calibrate: 1. Open the data file to calibrate. 2. Click the Spectrum window. 3. From the Process menu, select Mass Calibration, select Auto Calibrate State, then select On.
Automatic Calibration Applying new constants to the data file Calibration results To save the calibration constants for each spectrum in the data file, select Apply Calibration from the Process menu. Automatic calibration results are displayed in the Output window (Figure 5-9).
Chapter 5 Examining Spectrum Data 5.5 Centroiding NOTE: Centroiding is not supported for Mariner DAD data. To display peaks as centroid traces: 1. Click the Spectrum window to activate it. 2. Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing. 3. From the Process menu, select Centroiding. The centroid spectrum is displayed with a CT trace label (see Figure 5-10). The height of each vertical bar corresponds to the original peak area.
Mass Deconvolution (Mariner Data Only) 5.6 Mass Deconvolution (Mariner Data Only) NOTE: Mass deconvolution is not supported for Mariner DAD data. NOTE: The Mass Deconvolution software is an option in the Data Explorer software. The Multiple Charge command on the Process menu is dimmed if you have not purchased the option.
Chapter 5 Examining Spectrum Data The Multiply Charged Deconvolution dialog box (Figure 5-11) is displayed. 4. In the Spectrum window, right-click-drag one multiply charged peak. 5. Right-click-drag a second multiply charged peak adjacent to the first selected peak and in the same envelope of charged peaks. Mass/Charge values are entered in the list box.
Mass Deconvolution (Mariner Data Only) 6. Select the method to use for calculation: • Automatic—Selects additional multiply charged peaks based on the selected peaks and performs the calculation. NOTE: If the trace is noisy, the software may not accurately select additional multiply charged peaks. • Manual—Performs the calculation using only selected peaks. Does not select additional multiply charged peaks. 7. Select Apex or Centroid mass to use for the calculation. 8.
Chapter 5 Examining Spectrum Data 11. Click OK. The result is displayed in the Output window and the zero-charge spectrum is displayed with a DECONV trace label, if selected. NOTE: The numerical result displayed in the output window generally is more accurate than the computer-generated spectrum. 12. To return to the original spectrum, click in the toolbar. The number of the original spectrum is displayed in the Select Spectrum dialog box. 13. Click OK.
Mass Deconvolution (Mariner Data Only) 5. Type values for the following masses for the generated zero-charge spectrum: • Center—Center mass • Half Width—Mass (±) from the Center mass to include in the spectrum • Increment—Mass increment at which to perform the calculation (0.1 for resolved isotope peaks, 1.0 for unresolved isotope peaks, >1.0 for noisy trace) Figure 5-12 Zero-Charge Spectrum Conversion Dialog Box 6. Select or type the mass of the adduct ion to use in the calculation.
Chapter 5 Examining Spectrum Data 5.7 Noise Filtering/Smoothing Description The Noise Filter/Smooth function includes four options for reducing noise in a spectrum trace: • • • • Procedure Default smoothing Noise-filtering Smoothing by the Gaussian method Noise removal To noise-filter or smooth the display: 1. Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing. 2. From the Process menu, select Noise Filter/Smooth.
Noise Filtering/Smoothing 3. Type of Data Noisy, low-resolution data Select the method to use based on the type of data you are examining, then enter the associated value displayed for the method you select: Suggested Method Default smoothing (RSM) (May affect peak resolution.) Description No associated value is displayed.
Chapter 5 Examining Spectrum Data Type of Data High-resolution data Suggested Method Noise Removal (NR) (Does not affect peak resolution.) Description Specify the number of standard deviations of noise to remove. The software automatically calculates the average white noise for all frequencies across the spectrum, then removes the specified number of standard deviations of noise.
Adjusting the Baseline 5.8 Adjusting the Baseline This section includes: • Using Baseline Offset • Using Baseline Correction • Using Advanced Baseline Correction 5.8.1 Using Baseline Offset Use the Baseline Offset command to offset the y-axis in a spectrum or to correct a sloping baseline: 1. Activate the window in which you want to perform the offset. NOTE: You can select a Chromatogram or a Spectrum window.
Chapter 5 Examining Spectrum Data Figure 5-14 Baseline Offset Dialog Box 4. Right-click-drag the left baseline to offset. The selected value is displayed in the Left Baseline field. 5. Right-click-drag the right baseline to offset. The selected value is displayed in the Right Baseline field. 6. To limit the baseline offset to the area between the two selected points, select Only Apply from L to R Baseline Midpoint.
Adjusting the Baseline 5.8.2 Using Baseline Correction Description When to use The Baseline Correction function corrects for a curved baseline, including a DC-offset baseline, by eliminating broad artifacts from the data set. Baseline-correct if you are analyzing data: • With a baseline that is not flat, and you are using the %Base Peak Intensity parameter (intensity-based thresholding) to screen out noise peaks. For best results, apply Baseline Correction, then re-detect peaks.
Chapter 5 Examining Spectrum Data 5.8.
Adjusting the Baseline When to use Use advanced baseline correction if you are analyzing data with an offset in the spectrum, particularly data with a strong sloping baseline at low mass. NOTE: Because this function is iterative, it may take several seconds to complete, and typically takes longer for narrower peaks. Correcting the baseline To correct the baseline: 1. Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing. 2.
Chapter 5 Examining Spectrum Data 3. Enter parameters as described below. These parameters interact with each other and require experimentation to determine the optimum settings for your data. Refer to “General guidelines for setting parameters” on page 5-54 for more information. Parameter Peak Width (at half height) Description/Specifies Value the software uses to estimate the baseline amplitude at regularly spaced points in the spectrum.
Adjusting the Baseline Parameter Peak Width (at half height) Description/Specifies Set Peak Width according to the data you are correcting: • For best results, set to the peak width at half height of the narrowest peak. However, smaller peak width values increase processing time. • If peak width varies across the spectrum, set to the average peak width.
Chapter 5 Examining Spectrum Data Parameter Flexibility Description/Specifies With the Peak Width parameter, determines the number of points used to estimate the baseline amplitude at regularly spaced points in the spectrum, but is not directly proportional to the number of points used. Valid entries are 0 to 1.0. The default value of 0.5 works best for most applications. A value closer to 0 reduces flexibility and provides a smoother more generalized baseline correction (see below).
Adjusting the Baseline Parameter Degree Description/Specifies Determines how closely the calculated baseline fits the data. Valid entries are 0.0 to 1.0. A value closer to 1 fits the baseline more closely to the data, and corrects the midpoint of the noise signal to approximately 0 intensity. A value closer to 0 fits the baseline less closely to the data, and corrects the midpoint of the noise signal to a value greater than 0 intensity (see below).
Chapter 5 Examining Spectrum Data General guidelines for setting parameters Refer to the following table to determine how to set Advance Baseline Correction parameters and obtain the desired baseline correction. Condition Baseline follows a rising peak cluster Baseline is gently curving Baseline rise should be ignored and treated as signal Set Parameters To • Peak Width—Average peak width • Flexibility—0.5 • Degree—0.5 • Peak Width—10 to 20 times peak width • Flexibility—0 • Degree—0.
Adjusting the Baseline Troubleshooting If the baseline rises preceding and following a peak after the correction (a “hump” under the peak), adjust the following parameters in the order listed: • Decrease Flexibility • Decrease Degree • Decrease Peak Width NOTE: Lower Peak Width values increase the time needed for processing. Returning to the original spectrum To return to the original trace, see “Returning to the original spectrum” on page 5-3.
Chapter 5 Examining Spectrum Data 5.9 Truncating a Spectrum Description The Truncate function removes data points from a trace outside a selected region. Truncating spectra is useful to: • Mariner data—Remove noise at the low end of a spectrum before generating a result file • Voyager data—Eliminate the Low Mass Gate spike and background in the low-mass range Truncating To truncate spectra: 1. Display the spectrum to truncate. 2.
Truncating a Spectrum The data in the spectrum is truncated to the selected range and is displayed with a TR trace label. The range displayed on the axis of the truncated trace is the range of the original data file, and may be wider than the range of the truncated spectrum. 6. Example To return to the original trace, see “Returning to the original spectrum” on page 5-3. Figure 5-18 and Figure 5-19 illustrate the effects of truncating a Voyager spectrum and eliminating the Low Mass Gate peak.
Chapter 5 Examining Spectrum Data Figure 5-19 Truncated Spectrum—Low Mass Gate Spike Eliminated 5 5-58 Applied Biosystems
Converting to a Singly Charged Spectrum (Mariner Data Only) 5.10 Converting to a Singly Charged Spectrum (Mariner Data Only) NOTE: Single-charge conversion is not supported for Mariner DAD data. Description The Single-Charge Conversion function generates a theoretical, centroided, singly charged spectrum. This function uses isotopic spacing in detected spectral peaks to generate the theoretical spectrum.
Chapter 5 Examining Spectrum Data 3. Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing. 4. From the Process menu, select Single-Charge Conversion. The Single-Charge Spectrum Conversion dialog box (Figure 5-20) is displayed. Figure 5-20 Single-Charge Spectrum Conversion Dialog Box 5. In the Adduct text box, type or select the adduct that is the charge-carrying species in the spectrum you are examining. 6.
Converting to a Singly Charged Spectrum (Mariner Data Only) Example Figure 5-21 and Figure 5-22 illustrate the effects of single-charge conversion. Before conversion (Figure 5-21), the spectrum includes +2 and +3 charged species of neurotensin. Neurotensin multiply charged species Figure 5-21 Spectrum Before Single-Charge Conversion After conversion (Figure 5-22), the +2 and +3 charged species are converted to the +1 species of neurotensin.
Chapter 5 Examining Spectrum Data NOTE: Charge states other than 0 or 1 in the converted trace indicate that a peak in the original spectrum is labeled with an incorrect charge state. Set peak detection thresholds to disregard these peaks and convert the spectrum again. CAUTION A zero value in the Spec Peak list does not indicate a charge state of zero. It indicates that the software could not determine the charge state. 5.
AutoSaturation Correction (Mariner Data Only) Effect on Mariner .RST files The AutoSaturation Correction feature is not applied to .RST files saved from the Mariner Instrument Control Panel, even when Saturation Correction is turned on. Saturation requires information about the pulser frequency used to acquire the data, and this information is not stored in .RST files saved from the Instrument Control Panel.
Chapter 5 Examining Spectrum Data 5.12 Adding and Subtracting Raw or Processed Spectra from the Same or Different Data Files (Dual Spectral Trace Arithmetic) The Dual Spectral Trace Arithmetic function lets you add two spectra together, or subtract one spectrum from another. Spectra can be raw or processed, and can be from the same or different data files. To use the Dual Spectral Trace Arithmetic function: 1.
Adding and Subtracting Raw or Processed Spectra from the Same or Different Data Files (Dual Figure 5-23 Dual Spectral Trace Arithmetic Dialog Box 6. Set the Mass Tolerance within which data points from the different traces will be considered as the same mass. 7. Select Add or Subtract for Operation. 8. Select Add New Trace or Replace Active Trace for the result trace. 9. Click OK.
Chapter 5 Examining Spectrum Data 5 5-66 Applied Biosystems
Chapter 6 Using Tools and Applications 6 This chapter contains the following sections: 6.1 Using the Elemental Composition Calculator .......... 6-2 6.2 Using the Isotope Calculator ................ 6-13 6.3 Using the Mass Resolution Calculator .................. 6-20 6.4 Using the Signal-to-Noise Ratio Calculator ........... 6-23 6.5 Using the Ion Fragmentation Calculator ............... 6-25 6.6 Using the Elemental Targeting Application............ 6-31 6.7 Using the Macro Recorder ........
Chapter 6 Using Tools and Applications 6 6.1 Using the Elemental Composition Calculator This section includes: • • Determining elemental composition Setting limits 6.1.
Using the Elemental Composition Calculator Procedure To determine elemental composition: 1. Display the spectrum containing the peak of interest. 2. Click the Spectrum window to activate it. 3. From the Applications menu, select Elemental Composition. 6 The Elemental Composition Calculator dialog box (Figure 6-1) is displayed.
Chapter 6 Using Tools and Applications 4. 6 Enter m/z values in the m/z ratio list by doing any of the following: • Right-click-drag over a peak in the spectrum to add the m/z and the associated charge state. • Double-click the line to display the Elemental Target Mass dialog box, type the m/z and the associated charge state, then click OK.
Using the Elemental Composition Calculator 11. • “Adding new elements and setting limits” on page 6-9 • “Setting limits for other result types” on page 6-12 Click More Parameters, then enter the Minimum and Maximum Double Bond Equivalents to include in the calculation. Electron state for 12. Set the Electron State to calculate. If you are calculating composition for: intact molecules or fragment ions • Intact molecules—Use the default of Even Only.
Chapter 6 Using Tools and Applications 6 Hint: You can sort the results in a column by clicking the column header. Results include: • Index—Sequential number assigned to each result. • Input m/z—Entered m/z for each composition calculation. • Calculated Mass—Calculated mass for the entered m/z and charge state for each theoretical composition. • mDa Error and ppm Error—Error for each calculation.
Using the Elemental Composition Calculator Displaying the theoretical isotope distribution To display the theoretical isotope distribution for a calculated formula, double-click the corresponding line in the Elemental Analysis tab of the Output window. The theoretical isotope trace is displayed in the Spectrum window with an ISO trace label and the elemental formula. To return to the original trace, see “Returning to the original spectrum” on page 5-3. 6.1.
Chapter 6 Using Tools and Applications 2. 6 To change the limits for an element, double-click an element to display the Isotope dialog box (Figure 6-4). Figure 6-4 Isotope Dialog Box NOTE: Ignore the column of check boxes to the left of the Isotope column if it is displayed. 3. Change the Minimum and Maximum number of occurrences for the element as needed. NOTE: The software ignores changes you make to the individual isotope minimum and maximum values. 6-8 4. Click OK. 5.
Using the Elemental Composition Calculator Adding new elements and setting limits To add new elements and set limits: 6 1. To add new elements and set limits for Elemental results, click Element Limits in the Elemental Composition Calculator dialog box. The Limits dialog box is displayed (Figure 6-5). Figure 6-5 Element Limits Dialog Box 2. Click . The Periodic Table (Figure 6-6) is displayed.
Chapter 6 Using Tools and Applications 6 Figure 6-6 Periodic Table 3. 6-10 Applied Biosystems Click an element to select it and to display the Isotope dialog box (Figure 6-7).
Using the Elemental Composition Calculator 6 Figure 6-7 Isotope Dialog Box NOTE: Ignore the column of check boxes to the left of the Isotope column if it is displayed. 4. Change the Minimum and Maximum number of occurrences for the first isotope of the element as needed. NOTE: The software ignores changes you make to the individual isotope minimum and maximum values. 5. Click OK two times to return to the Element Limits dialog box. 6.
Chapter 6 6 Using Tools and Applications Setting limits for other result types To set limits for amino acid, DNA, RNA, or carbohydrate result types: 1. Click the limits button displayed for the selected result type in the Elemental Composition Calculator dialog box (see Figure 6-1 on page 6-3). The Limits dialog box for the selected Result Type is displayed. Figure 6-8 shows the Amino Acid Limits dialog box. Figure 6-8 Amino Acid Limits Dialog Box 6-12 Applied Biosystems 2.
Using the Isotope Calculator 6.2 Using the Isotope Calculator Description Using the Isotope Calculator 6 Use the Isotope calculator to generate a theoretical isotope distribution. You can compare or overlay the theoretical distribution with your observed distribution. To use the Isotope calculator: 1. Display the spectrum containing the observed isotope distribution. 2. Click the Spectrum window to activate it. 3. From the Applications menu, select Isotope Calculator.
Chapter 6 Using Tools and Applications 6 4. Select the Formula or Sequence for the type of isotope to calculate. 5. Select a formula from the list, or type in a new formula. Valid entries for each formula type are: Formula Type Valid Entries Elemental Any element from the Periodic Table. The first letter of two-letter elemental symbols must be capitalized. Spaces do not matter. Amino Acid Sequence One-letter or three-letter amino acid codes.
Using the Isotope Calculator 7. 8. Specify the Add/Subtract Group option. To enable or disable the option, select or deselect the Group Type check box. 6 In the Add/Subtract Group section, select: • Element or group to add or subtract from the formula before calculating the isotope (does not apply if Add/Subtract Group is disabled). • Number of elements or groups to add or remove and the charge state to divide by.
Chapter 6 Using Tools and Applications 10. 6 Select the calculation mode: • FWHM—Resolves peaks using the full peak width at peak half height. • 10% Valley—Resolves peaks to a 10 percent valley. 11. Set the Threshold %. Signal intensity below this percent intensity is not included in the calculation or display. NOTE: Setting Threshold % too high distorts the isotope pattern. 12. Click Calculate.
Using the Isotope Calculator To calculate a theoretical isotope distribution for doubly sodiated and deprotonated β-cyclodextrin with –1 charge state (m/z –1089) With these parameters specified Formula*—C42H60O30Na2 The software performs this calculation (C42H60O30Na2 – H) Group type—H Group Count/Charge—1 Subtract To calculate a theoretical isotope distribution for β-cyclodextrin with +2 charge state (m/z 522) With these parameters specified Formula*—C42H60O30 The software performs this calculation C42
Chapter 6 6 Using Tools and Applications Evaluating traces The theoretical isotope distribution is displayed in the Spectrum window with an ISO trace label (Figure 6-10). Figure 6-10 Isotope Trace If you have the Replace mode set to Add in the Display Trace dialog box, a new trace is added. If you have the Replace mode set to Replace, the isotope trace replaces the active trace. For more information on Replace mode, see “Setting the Replace mode” on page 2-17.
Using the Isotope Calculator Results The results of the calculation are displayed in the Result tab of Output window (Figure 6-11). Figure 6-11 Isotope Calculation Results in Output Window If results are not calculated If results are not calculated, you may have tried to remove a group that is not present in the formula.
Chapter 6 Using Tools and Applications 6 6.3 Using the Mass Resolution Calculator Calculating mass resolution To calculate mass resolution: 1. Display the spectrum of interest. Make sure the Spectrum window is active. 2. If you are examining Voyager data, baseline correct the spectrum. The Peak Height used by the Resolution calculator is calculated from 0. For information, see Section 5.8.2, Using Baseline Correction. NOTE: Baselines in Mariner data are typically at 0.
Using the Mass Resolution Calculator 6 FWHM=50% peak height Figure 6-12 Mass Resolution Calculator 5. Specify the peak for which to calculate resolution by doing one of the following: • Type in a Mass/Charge value. • In the Spectrum window, right-click-drag over the peak for which you want to calculate resolution. The mass is automatically entered in the Resolution Calculator dialog box. NOTE: If you right-click-drag over more than one peak, the mass of the highest peak is used. 6. Click OK.
Chapter 6 Using Tools and Applications The result is displayed in the Output window (Figure 6-13).
Using the Signal-to-Noise Ratio Calculator 6.4 Using the Signal-to-Noise Ratio Calculator Description A signal-to-noise ratio is typically used to describe how well a peak of interest in a spectrum or chromatogram is distinguished from background noise. The Data Explorer software provides two signal-to-noise ratio methods that calculate signal-to-RMS noise (electronic and chemical): • Auto—You specify the peak for calculation and the software automatically calculates the average noise across the spectrum.
Chapter 6 Using Tools and Applications 3. 6 Select the method to use, then right-click-drag on peaks in the trace to enter the associated values displayed for the method you select: • Auto—Right-click-drag across the apex of the peak for signal-to-noise calculation. The software automatically calculates the average noise across the entire spectrum. The noise calculation is not affected by the presence of peaks or a poor baseline.
Using the Ion Fragmentation Calculator 6.5 Using the Ion Fragmentation Calculator Description 6 The Ion Fragmentation calculator generates a list of possible fragment masses for a peptide sequence you enter. It can calculate the masses for: • Multiply charged fragments you may see in Mariner data. • PSD fragments you may see in Voyager data. If the calculated fragments are present in the current data file, you can label fragments.
Chapter 6 Using Tools and Applications 6 Figure 6-15 Ion Fragmentation Calculator Dialog Box 4. Type or copy the amino acid or residue sequence of interest. Use single-letter codes. Sequence codes are case-sensitive. Click the User-Defined Amino Acids button to display the list of allowed residues and their associated codes. 6-26 Applied Biosystems 5. Select the N-terminus and C-terminus options for the sequence. 6.
Using the Ion Fragmentation Calculator Setting Options 7. Click Options. The Ion Fragmentation Options dialog box (Figure 6-16) is displayed. Figure 6-16 Ion Fragmentation Options Dialog Box 8. Select the ion types to display, and whether or not to generate results for internal fragments. 9. If you are analyzing Mariner data, select Calculate multiple charge states if desired, then specify the maximum charge state to calculate (up to 12). 10. Type the Mass Tolerance to use when labeling peaks.
Chapter 6 6 Using Tools and Applications Setting 13. Click User-Defined Amino Acids. user-defined amino The User-Defined Amino Acids dialog box (Figure 6-17) acids is displayed. Figure 6-17 User-Defined Amino Acids Dialog Box 14. Add amino acid definitions and codes as needed. NOTE: You cannot modify pre-defined amino acids. User-defined amino acids are not saved when you close the Data Explorer software. 15. Click Close. Calculating the 16. Click Induce Fragmentation.
Using the Ion Fragmentation Calculator 6 Figure 6-18 Ion Fragmentation Results for Synthetic Peptide (PPPPPPPPPPPPAR) Results Results are displayed in the: • Ions table—Lists the masses for each fragment and ion type. • Internal fragments table—Lists possible internal sequences, if you enabled this option in the Options dialog box (see Figure 6-16 on page 6-27). Click Clear Table Info to clear results. You can change options and recalculate ion fragmentation results.
Chapter 6 Using Tools and Applications Labeling peaks 6 Click Label Peaks. The ion peaks specified in the Options dialog box are labeled on the trace if they are present (Figure 6-19). Hint: To screen out labels, decrease the Label Tolerance in the Options dialog box. Figure 6-19 Labeled Ion Fragmentation Peaks for Synthetic Peptide (PPPPPPPPPPPPAR) Hint: The Label Peaks function creates User Labels in the data file. To view, select Peak Label from the Peaks menu, then select User Label Setup.
Using the Elemental Targeting Application 6.6 Using the Elemental Targeting Application Description 6 The Elemental Targeting application determines if observed masses in a spectrum correspond to chemical formulas you enter. This application generates a theoretical isotope pattern for the mass you enter using the Mass Resolution specified in Basic Peak Detection settings.
Chapter 6 Using Tools and Applications Note the following when entering formulas: 6 • Spaces do not matter for formula. The first letter of two-letter elemental symbols must be capitalized (for example, Na). • To ensure a better match between theoretical and observed isotopes, include the appropriate number of protons in the formula you enter for multiply charged ions. Figure 6-20 Elemental Targeting Dialog Box 7. 6-32 Applied Biosystems Click Calculate.
Using the Elemental Targeting Application Displaying results The results of the calculation are displayed in the Elemental Target tab of the Output window (Figure 6-21). Figure 6-21 Elemental Targeting Results in the Output Window Results include: • Index—Sequential number assigned to each result. • Formula—Elemental composition you entered. • m/z—Mass/charge of an observed peak that, compared to the theoretical mass of the formula specified, is within the Mass Tolerance and Resolution you specified.
Chapter 6 Using Tools and Applications 6 6.7 Using the Macro Recorder Description Macros provided In this section The Macro Recorder feature in Data Explorer allows you to set up multi-step tasks to execute automatically when you click a macro button. The Macro Recorder feature creates a Visual Basic script of your actions as you record the macro, then executes the script when you run the macro.
Using the Macro Recorder Location of macros Displaying the macro toolbar All macros you record are stored in a file called DATAEXPLORER.VB6 in the C:\MARINER\PROGRAM or C:\VOYAGER directory. If the macro toolbar (Figure 6-22) is not displayed: 1. Select Toolbar from the View menu. 2. Select Macros, then click Close. Figure 6-22 Macro Toolbar If a numbered macro button is disabled (gray), no macro has been assigned to it. If a numbered macro button is enabled (green), a macro has been assigned to it.
Chapter 6 6 Using Tools and Applications Menu View Commands Not Supported No commands supported Display • Add/Remove Traces NOTE: The and buttons are supported by the Macro Editor.
Using the Macro Recorder 6.7.2 Recording a Macro 6 To record a macro: 1. Open a data file. 2. From the Tools menu, select Record New Macro. The Record Macro dialog box (Figure 6-23) is displayed. Figure 6-23 Record Macro Dialog Box 3. Type a name and a description if desired. 4. Click OK. 5. Select the commands you want to automate with the macro. For example, select Noise Filter/Smooth from the Process menu, select the smoothing method, specify the associated parameter, then click OK.
Chapter 6 6 Using Tools and Applications 6.7.3 Assigning Macros to Buttons Only macros present in the DATAEXPLORER.VB6 file can be assigned to buttons and run in the Data Explorer software. NOTE: If you have installed a new version of Data Explorer software, new macros may be provided. New macros are not available for use until you import them into the Data Explorer project. For information, see Section 6.7.7, Importing or Exporting Macros in DATAEXPLORER.VB6.
Using the Macro Recorder De-assigning a macro from a button To de-assign a macro from a button, select the macro button in the Assign Macro dialog box, then click De-assign. 6.7.4 Running a Macro You can run a macro using a: • • Using a toolbar button Toolbar button Menu command To run a macro using the toolbar button: 1. Open the data file on which you want to run the macro. 2. Click the button assigned to the macro you want to run.
Chapter 6 Using Tools and Applications 6 Figure 6-25 Macros Dialog Box 3. Select the macro to run from the list. 4. Click Run. The macro executes. If the macro contains a syntax error If the macro contains a syntax error, it may cause the Data Explorer software to close unexpectedly. If this occurs, restart the Data Explorer software and examine the macro in the Visual Basic Editor. See Section 6.7.6, Advanced Macro Editing.
Using the Macro Recorder 6.7.5 Deleting a Macro 6 To delete a macro: 1. From the Tools menu, select Macros. The Macros dialog box (Figure 6-26) is displayed. Figure 6-26 Macros Dialog Box 2. Select the macro to delete from the list. 3. Click Delete. NOTE: Other buttons on this dialog box are for advanced editing. Refer to the online help available within the Visual Basic Editor.
Chapter 6 6 Using Tools and Applications 6.7.6 Advanced Macro Editing Accessing the Visual Basic Editor You can access the Visual Basic Editor to enhance or edit a script created by the Macro Recorder in Data Explorer, or to create a new script. Access the Visual Basic Editor from Data Explorer in three ways: • Select Visual Basic Editor from the Tools menu. • Select Macros from the Tools menu, select the macro to edit, then click Edit. • Click in the Macro toolbar The DATAEXPLORER.
Using the Macro Recorder 6.7.7 Importing or Exporting Macros in DATAEXPLORER.VB6 6 You can import macros into, or export macros from, the DATAEXPLORER.VB6 project for use in the Data Explorer software. Importing when new versions of Data Explorer software installed When you install a new version of the Data Explorer software: • The DATAEXPLORER.VB6 file is not overwritten. This allows you to maintain any macros you have developed in the DATAEXPLORER.VB6 file.
Chapter 6 Using Tools and Applications The selected macros are imported into the DATAEXPLORER.VB6 project. The .BAS files are included in the Modules folder in the DataExplorerProject, and the .FRM files are included in the Forms folder in the DataExplorerProject. All macros imported into the DataExplorerProject are displayed in the list of macros you can assign in the Data Explorer software. See Section 6.7.3, Assigning Macros to Buttons. 6 Exporting To export macros: 1.
Using the Macro Recorder 6.7.8 Running Macros Automatically When Opening and Closing Files 6 You can set the Data Explorer software to automatically run macros you previously created when you open or close a data file. For information on creating macros, see Section 6.7.2, Recording a Macro. To set up automatic macros: 1. From the Tools menu, select Automatic Macros. The Automatic Macro Setup dialog box (Figure 6-26) is displayed. Figure 6-27 Automatic Macro Setup Dialog Box 2.
Chapter 6 Using Tools and Applications 6 6-46 Applied Biosystems
Chapter 7 Data Explorer Examples 7 This chapter contains the following sections: 7.1 7.2 Mariner Data Examples ...................................... 7-2 7.1.1 Improving Signal-To-Noise Ratio ......... 7-2 7.1.2 Deconvoluting and Evaluating Unresolved Chromatographic Peaks ... 7-4 7.1.3 Determining if a Peak is Background Noise .............................. 7-8 Voyager Data Examples..................................... 7-11 7.2.1 Detecting and Labeling Partially Resolved Peaks ..................
Chapter 7 Data Explorer Examples 7.1 Mariner Data Examples This section includes: • Improving signal-to-noise ratio • Deconvoluting and evaluating unresolved chromatographic peaks • Determining if a peak is background noise 7 7.1.1 Improving Signal-To-Noise Ratio Overview You can improve the signal-to-noise ratio for low-level components in the total ion chromatogram (TIC) by creating an extracted ion chromatogram for the mass of interest.
Mariner Data Examples Figure 7-1 illustrates the improved signal-to-noise ratio in the extracted ion chromatogram for three replicate loop injections.
Chapter 7 Data Explorer Examples 7.1.
Mariner Data Examples The combined spectrum is displayed (Figure 7-3), with two intense peaks at 410 Da and 723 Da. Generate extracted ion chromatograms as described below to determine if these peaks are the coeluting components. NOTE: If these peaks do not yield chromatograms with profiles that correspond to the unresolved peaks, try another spectral peak. 7 Figure 7-3 Combined Spectrum for Unresolved Peaks in Cytochrome C Creating extracted ion chromatograms To create extracted ion chromatograms: 1.
Chapter 7 Data Explorer Examples 7 Original TIC containing unresolved peaks Extracted ion chromatograms with deconvoluted peaks Figure 7-4 Deconvoluting Unresolved Chromatographic Peaks Creating combined spectra 7-6 Applied Biosystems Create a combined spectrum for each extracted ion chromatogram: 1. Activate the Spectrum window, then click toolbar two times to add two traces. in the 2. Right-click-drag over the first half of the mass range in the 410 extracted ion chromatogram. 3.
Mariner Data Examples Figure 7-5 illustrates the combined spectra for the deconvoluted peaks. Note that both spectra contain a peak at 391 Da which requires investigation to determine if it is a low-level component or background noise. See Section 7.1.3, Determining if a Peak is Background Noise.
Chapter 7 Data Explorer Examples 7.1.3 Determining if a Peak is Background Noise Overview To determine if spectral peaks represent low-level components or if they are due to solvent contribution, you can: 7 Subtracting spectral peaks • Subtract the spectral peaks from the chromatogram • Create an extracted ion chromatogram for the spectral peaks The following example uses the chromatograms and spectra from Section 7.1.2, Deconvoluting and Evaluating Unresolved Chromatographic Peaks.
Mariner Data Examples The spectrum range is displayed in the Spectra To Be Subtracted list in the Add and Subtract Spectra dialog box (Figure 7-6). 7 Figure 7-6 Subtracting Spectra 6. Click OK. The subtracted spectrum is displayed (Figure 7-7 on page 7-10).
Chapter 7 Data Explorer Examples Figure 7-7 Subtracted Spectrum 7 The peak at 391 is still present, which indicates one of the following conditions: • • You did not subtract sufficient baseline The peak is a coeluting component To evaluate further, create an extracted ion chromatogram for 391 Da as described below and evaluate the signal. Creating extracted ion chromatogram To create the extracted ion chromatogram, right-click-drag over the peak at 391 Da in the spectrum trace.
Voyager Data Examples 7.2 Voyager Data Examples This section includes: • Detecting and labeling partially resolved peaks • Processing before calibrating to optimize mass accuracy • Detecting peaks from complex digests 7 7.2.1 Detecting and Labeling Partially Resolved Peaks If peaks are not labeled If peaks are partially resolved and the peaks of interest are not labeled, you can adjust the following peak detection parameters: If... Adjust the following...
Chapter 7 7 Data Explorer Examples Figure 7-9 Partially Resolved Peaks That Represent Two Compounds, Minor Component Not Detected Adjusting peak detection To adjust peak detection: 1. Click in the toolbar or select Peak Detection from the Peaks menu. The Spectrum Peak Detection Setup dialog box is displayed with the Basic Settings tab (Figure 7-10) displayed.
Voyager Data Examples 2. Do either of the following: • Change the %Max Peak Area from 1 (the default) to 0, and the %Base Peak Intensity from 0 (the default) to 1. • Click the Peak Processing tab and change the default Integration Baseline Setting from Valley-to-Valley to Valley-to-Baseline. 3. Click OK. Figure 7-11 shows the partially resolved peaks that are now labeled.
Chapter 7 Data Explorer Examples 7.2.2 Processing Before Calibrating to Optimize Mass Accuracy This section includes: • Calibrating without baseline correcting and deisotoping • Before calibrating • Calibrating 7 Calibrating without baseline correcting and deisotoping For optimum mass accuracy, baseline correct and deisotope a spectrum before calibrating. Figure 7-12 shows a spectrum before calibration.
Voyager Data Examples Before calibrating Baseline correcting To optimize mass accuracy, do the following before calibrating: 1. Display the spectrum of interest. 2. From the Process menu, select Baseline Correction. The spectrum is baseline corrected. For more information, see Section 5.8.2, Using Baseline Correction. Deisotoping 3. From the Peaks menu, select Peak Deisotoping. The Deisotoping dialog box (Figure 7-14) is displayed. Figure 7-14 Deisotoping Dialog Box 4.
Chapter 7 Data Explorer Examples Calibrating To calibrate the deisotoped spectrum: 1. From the Peaks menu, select Peak Label, and select the Mass Label Type (peak apex or peak centroid) to use for calibration. Click OK. 2. From the Process menu, select Mass Calibration and then select Manual Calibration. The Manual Calibration dialog box is displayed (Figure 7-16). 7 Figure 7-16 Manual Calibration Dialog Box 3. Click , then select the VOYAGER.REF calibration reference file. 4.
Voyager Data Examples Matching peaks 5. Click Match Peaks and Solve. The software compares observed masses in the spectrum to reference masses in the selected reference file, lists the matches in the Peak Matched list, calibrates the spectrum, and displays the calibration statistics in the Output window. NOTE: If you set Mass Tolerance too low, no peaks will match.
Chapter 7 Data Explorer Examples 7.2.3 Detecting Peaks from Complex Digests Overview Complex digests often contain hundreds of peaks which may have relatively low signal-to-noise ratios. To quickly screen out noise and detect peaks of interest: • Noise filter/smooth to remove initial noise. 7 • Set initial peak detection thresholds low enough to detect all peaks, to ensure that monoisotopic peaks are detected for proper deisotoping. • Deisotope to identify isotope peak clusters.
Voyager Data Examples Procedure Noise filtering/smoothing To detect peaks from complex mixtures: 1. Display the spectrum of interest. 2. From the Process menu, select Noise Filter/Smooth. The Noise Filter/Smooth dialog box (Figure 7-19) is displayed. 7 Figure 7-19 Noise Filter/Smooth Dialog Box 3. Select Default Smoothing or Noise Filter (with a Correlation Factor of 0.7), then click OK. For more information, see Section 5.7, Noise Filtering/Smoothing. Setting detection thresholds 4.
Chapter 7 Data Explorer Examples 7 Figure 7-20 Spectrum Peak Detection Setup— Basic Settings Tab 5. Click Use Advanced Settings. The Advanced Settings tab is displayed (Figure 7-21).
Voyager Data Examples 7 Figure 7-21 Spectrum Peak Detection Setup— Advanced Settings Tab 6. Set Minimum Area to 0. 7. Click the Basic Settings tab (see Figure 7-20 on page 7-20), then set %Max Peak Area to 0 or 0.1. 8. Click Apply. Note that many peaks are added to the peak list in the Output window. For more information, see Section 3.2, Peak Detection. Deisotoping 9. From the Peaks menu, select Peak Deisotoping. The Deisotoping dialog box (Figure 7-14) is displayed.
Chapter 7 Data Explorer Examples 7 Figure 7-22 Deisotoping Dialog Box 10. For this example spectrum, specify H for Adduct and C6H5NO for Generic Formula. 11. Click OK. For more information on deisotoping, see Section 3.4, Deisotoping a Spectrum. Increasing 12. In the Basic Settings tab in Global Thresholds (see Figure 7-20 on page 7-20), increase %Max Peak Area detection to a setting between 0.1 and 1% until the peaks of thresholds interest are detected and noise is screened out.
Chapter 8 Viewing Voyager PSD Data 8 This chapter contains the following sections: 8.1 Displaying PSD Data ....................................... 8-2 8.2 Applying Fragment Labels ............................... 8-8 8.3 Calibrating a PSD Spectrum .......................... 8-10 8.3.1 Checking Peak Detection ............... 8-11 8.3.2 Calibrating ..................................... 8-12 8.3.3 Creating PSD .CAL Files and Applying to Other Data Files .......... 8-20 8.3.
Chapter 8 Viewing Voyager PSD Data 8.1 Displaying PSD Data This section includes: • • • • • Displaying the composite spectrum Advancing through segment traces Displaying multiple segment traces Redisplaying the composite spectrum How the composite spectrum is generated NOTE: For information on acquiring PSD spectra, see the Voyager Biospectrometry Workstation User’s Guide.
Displaying PSD Data 8 Figure 8-1 PSD Spectrum in Data Explorer Advancing through segment traces To advance through segment traces, click and . Segments are displayed in the order in which they were acquired. NOTE: If these buttons are not displayed in the toolbar, you can add them. See Section 1.4.3, Customizing Toolbars. Displaying multiple segment traces To display multiple segment traces: 1. Click in the toolbar three times to add three “Not Used” traces. 2.
Chapter 8 Viewing Voyager PSD Data The PSD Processing dialog box is displayed (Figure 8-2) and lists all segments contained in the PSD .DAT file in the order in which they were acquired with associated Mirror Ratios and Max Stitch Masses. Column header 8 Figure 8-2 PSD Processing Dialog Box The Max Stitch Mass is equal to the Precursor Mass times the Mirror Ratio. This value reflects the maximum mass of the segment that will be included in the composite spectrum.
Displaying PSD Data NOTE: The entry number in the PSD Segment list above may not correspond to Segment number specified in the Segment list for acquisition (described in the Voyager Biospectrometry Workstation User’s Guide) if you did not acquire all segments, or did not acquire segments in order of decreasing Mirror Ratio. The number above reflects the order of acquisition.
Chapter 8 Viewing Voyager PSD Data How the composite spectrum is generated The software does the following to generate a composite spectrum: • Evaluates all segments in the .DAT file to determine if there are multiple segments acquired using the same PSD Mirror Ratio. • If there are duplicates, selects the most recently acquired segment to include in the composite spectrum. • Using the precursor ion mass and calibration constants from the PSD calibration in the .
Displaying PSD Data If you are performing an internal standard calibration, the software determines the constants as listed below: PSD Internal Constant Standard Calibration One-point Two-point or three-point More than three-point Region of segments included in composite spectrum Value Used α Calculated from standard mass β and γ 0 α and β Calculated from standard masses γ 0 α, β, γ Calculated from standard masses The composite spectrum is generated from portions of the segment traces.
Chapter 8 Viewing Voyager PSD Data 8.2 Applying Fragment Labels Overview Use the Ion Fragmentation calculator to apply fragment labels. For detailed information on using the Ion Fragmentation calculator, see Section 6.5, Using the Ion Fragmentation Calculator. Applying labels To apply fragment labels to PSD spectra: 1. From the Applications menu, select Ion Fragmentation Calculator. The Ion Fragmentation Calculator dialog box (Figure 8-4) is displayed.
Applying Fragment Labels 2. In the Sequence text box, type the amino acid sequence of the compound. Use single-letter codes. Set other parameters as needed. For parameter descriptions, see Section 6.5, Using the Ion Fragmentation Calculator. 3. Click Options to specify the fragment peaks to label. 4. Click Induce Fragmentation. 5. Click Label Peaks. The ion peaks specified in Options are labeled on the trace if they are present.
Chapter 8 Viewing Voyager PSD Data 8.3 Calibrating a PSD Spectrum NOTE: Multi-point calibration yields higher mass accuracy than one-point calibration. This section includes: • • • • • When to use this procedure 8 Checking peak detection Calibrating Creating PSD .CAL files and applying to other data files Creating PSD calibration reference (.REF) files Changing the precursor mass Use this procedure to: • Generate a PSD calibration (.CAL) file from a known standard analyzed in PSD mode.
Calibrating a PSD Spectrum 8.3.1 Checking Peak Detection Checking Before calibrating, check that peaks in all segment traces of interest are properly peak detected and that noise is not detected as peaks. Note the following when setting peak detection parameters: • Peak detection settings are applied to the currently displayed composite spectrum or segment traces. • The default peak detection settings (in VOYAGERPSD.SET) have different detection ranges for different mass ranges.
Chapter 8 Viewing Voyager PSD Data 8.3.2 Calibrating This section includes: • • • • • • Calibrating Calibrating Matching peaks automatically Selecting peaks manually Solving and plotting Applying new constants to the data file Selecting calibration peaks for optimum mass accuracy To calibrate a PSD spectrum: 1. Click the Spectrum window to activate it. Select the spectrum of interest.
Calibrating a PSD Spectrum Column header buttons 8 Figure 8-5 PSD Processing Dialog Box with Calibration Tab Displayed 4. Select a PSD Calibration Reference File that you generated as described in Section 8.3.4, Creating PSD Calibration Reference (.REF) Files. A calibration reference file called Angiotensin_Fragments.REF is provided with the software. NOTE: Use a calibration reference (.
Chapter 8 Viewing Voyager PSD Data NOTE: If the calibration reference file is stored on a network drive, an error message may display when you select the calibration file when performing a calibration. If an error message is displayed, copy the file to a local drive on your computer using Windows NT Explorer. 5. Enter Reference Matching Criteria: • Minimum Intensity—Peaks must be above this intensity to be considered a match. Select the unit for Minimum Intensity, % Relative Intensity or % Relative Area.
Calibrating a PSD Spectrum Matching peaks automatically If you want the software to compare observed masses in all segment spectra included in the .DAT file to reference masses in the selected calibration reference file: 1. Click Match. If a mass within the tolerance of any of the masses listed in the calibration reference file is found in any spectrum in the .DAT file, the match is displayed in the Calibration Mass Peak Selection window. CAUTION Use the Match function with care.
Chapter 8 Viewing Voyager PSD Data Selecting peaks manually For optimum mass accuracy, select peaks as described in “Selecting calibration peaks for optimum mass accuracy” on page 8-19. To manually select the reference mass for a peak: 1. Right-click-drag over the peak of interest. The Reference Mass dialog box (Figure 8-6) is displayed and lists all masses in the calibration reference file.
Calibrating a PSD Spectrum 2. Do any of the following: • Click OK to accept the highlighted reference mass for matching. • Select a different reference mass and click OK. • Type new reference mass information in the Name, Theoretical Mass, Charge and Elemental Composition text boxes and select the mass type. Click OK to accept the reference mass for matching. NOTE: You must type in a minus sign (-) for negative charge states.
Chapter 8 Viewing Voyager PSD Data Solving and plotting After matching peaks, click Solve and Plot. The calibration statistics are displayed in the Result tab of the Output window and the calibration constants are applied to the spectrum displayed If you calibrate more than one time, subsequent calibration statistics are added to the end of the list in the Output window. Older calibration statistics are listed at the top of the list. Use the scroll bar to view newer statistics at the bottom of the list.
Calibrating a PSD Spectrum Selecting calibration peaks for optimum mass accuracy To improve calibration statistics, you can select the same fragment ion from more than one segment. Monoamino acid fragments (immonium ions) below 150 Da are useful for this purpose. Because the segments have been collected with different PSD Mirror Ratios, the software allows you to add the same mass to the list multiple times. Hint: A calibration reference file called Immonium_Ions.REF is provided with the software.
Chapter 8 Viewing Voyager PSD Data 8.3.3 Creating PSD Calibration (.CAL) Files and Applying to Other Data Files Creating PSD .CAL files To generate a PSD .CAL file: 1. Acquire a standard, for example, angiotensin, in the Instrument Control Panel in PSD mode. For more information, see the Voyager Biospectrometry Workstation User’s Guide. 2. Open the .DAT file in the Data Explorer software. 3. Calibrate as described in Section 8.3.2, Calibrating. 4.
Calibrating a PSD Spectrum 8.3.4 Creating PSD Calibration Reference (.REF) Files Overview You can manually create a calibration reference file by typing masses in a text file as described in Section 5.3.3, Creating or Modifying a Calibration Reference File (.REF). You can also use the Ion Fragmentation calculator to generate theoretical fragments and masses from the sequence for a standard compound, then automatically save the masses and associated information in a calibration reference file.
Chapter 8 Viewing Voyager PSD Data NOTE: This selection determines the mass type specified for the reference masses in the calibration reference file. Use a calibration reference (.REF) file that specifies the peak type for reference masses as Resolved Isotope Mass (even if they are not resolved isotopes). The calibration routine checks peak width to determine if a peak matches a Resolved Isotope Mass or an Average mass.
Calibrating a PSD Spectrum 8.3.5 Changing the Precursor Mass When to change precursor mass When analyzing the composite spectrum, you may find that the observed fragments and sequence are not consistent with the precursor mass used to acquire the .DAT file. For example, you acquired the data with a precursor mass of 1,000.5 Da, and while examining the data in Data Explorer, you realize that the data may correspond to a precursor mass of 1,000 Da or 1,001 Da.
Chapter 8 Viewing Voyager PSD Data Changing If the precursor mass taken from the data file is not correct: 1. Display the Segments tab (see Figure 8-2 on page 8-4) by doing either of the following: • In the PSD Calibration dialog box, click the Segments tab • From the Process menu, select PSD Processing 2. Click Change Mass and enter a new mass to use for calibration. 3. Click OK. 4. Click Plot to display the composite spectrum for the new mass.
Chapter 9 9 Troubleshooting This chapter contains the following sections: 9.1 Overview ......................................................... 9-2 9.2 General Troubleshooting.................................. 9-3 9.3 Processing, Tools, and Applications Troubleshooting ............................................... 9-6 9.4 Calibration Troubleshooting ........................... 9-10 9.5 Printing Troubleshooting ................................ 9-14 9.
Chapter 9 Troubleshooting 9.1 Overview This section includes: • • • • • General troubleshooting Processing, tools, and applications troubleshooting Calibration troubleshooting Printing troubleshooting Peak detection and labeling troubleshooting Troubleshooting information is organized according to likelihood of possible cause, from most likely to least likely possible cause. If you are unable to solve your problem using the information in the following tables, call Applied Biosystems Technical Support.
General Troubleshooting 9.2 General Troubleshooting Table 9-1 General Troubleshooting—Mariner and Voyager Symptom Possible Cause Action Cannot find data file Did not save the spectrum to a .DAT file Reacquire spectrum. Save .DAT file. Error message displayed when opening PSD data file You are currently acquiring the PSD DAT file and have not stopped the experiment Stop the Experiment. See the Voyager Biospectrometry Workstation User’s Guide.
Chapter 9 Troubleshooting Table 9-1 General Troubleshooting—Mariner and Voyager (Continued) Symptom Possible Cause Action M/z range in data files converted to centroid does not match m/z range in original data file M/z range in a data file that is converted from profile to centroid is determined by the peak detection range set in Data Explorer, not the m/z range in the original data file No action. Normal occurrence.
General Troubleshooting Table 9-2 General Troubleshooting—Mariner Only (Continued) Symptom Possible Cause Action Spectra labeled with spectrum numbers that do not correspond to the axis You are viewing event-filtered MS Method data. Spectra in an event-filtered trace are numbered contiguously (1,2,3...) regardless of their relation to the overall acquisition. However, the axis of the trace reflects the numbering of the overall experiment. No action. Normal occurrence. Conversion of .SPC to .
Chapter 9 Troubleshooting 9.3 Processing, Tools, and Applications Troubleshooting Table 9-4 Processing, Tools, and Applications Troubleshooting—Mariner and Voyager Symptom Possible Cause Action Failed to calculate result for isotope calculator You may have tried to remove a group that is not present in the formula Can only remove a group that is present in the formula. For information, see Section 6.2, Using the Isotope Calculator.
Processing, Tools, and Applications Troubleshooting Table 9-4 Processing, Tools, and Applications Troubleshooting—Mariner and Voyager (Continued) Symptom Possible Cause Action Results not saved for all traces in an overlaid trace Only results for the active trace are saved Display individual traces, then save results for each trace.
Chapter 9 Troubleshooting Table 9-4 Processing, Tools, and Applications Troubleshooting—Mariner and Voyager (Continued) Symptom Possible Cause Action After Single-charge Conversion of multiply charged peaks, you see charge states other than 0 or 1 Peaks in the original spectrum are labeled with an incorrect charge state 1. Set peak detection thresholds to disregard these peaks. See Section 3.2.5, Charge State Determination and Examples. 2. Convert the spectrum again. See Section 5.
Processing, Tools, and Applications Troubleshooting Table 9-5 Processing, Tools, and Applications Troubleshooting—Mariner Only Symptom Possible Cause Action Failed to calculate result for mass deconvolution Did not select at least two peaks for same charge envelope Select at least two peaks (for example, +1 and +2). For more information, see Section 5.6, Mass Deconvolution (Mariner Data Only).
Chapter 9 Troubleshooting 9.4 Calibration Troubleshooting Table 9-6 Calibration Troubleshooting—Mariner and Voyager Symptom Possible Cause Action Auto Calibration is turned on, but current spectrum is not auto calibrated Current spectrum is not calibrated until the next time the spectrum is displayed Advance to the next spectrum, then return to the current spectrum. For more information, see Section 5.4, Automatic Calibration.
Calibration Troubleshooting Table 9-6 Calibration Troubleshooting—Mariner and Voyager (Continued) Symptom Possible Cause Action Calibration returns an invalid number of matches When creating a reference mass list in Manual or Automatic calibration, the software allows you to add multiple items with the same m/z value to the calibration list box if any other attribute of the reference compound is different (for example, charge state or name). Each mass in the list is considered during calibration.
Chapter 9 Troubleshooting Table 9-7 Calibration Troubleshooting—Mariner Only Symptom Possible Cause Action Mass Calibration commands are dimmed Chromatogram window is selected Select Spectrum window. Apply Calibration command is dimmed when calibrating MS Method data For MS Method data, calibration is valid for an individual spectrum. You cannot apply the calibration from one spectrum to the entire data file. No action. Normal occurrence. Error displayed when you import a calibration .
Calibration Troubleshooting Table 9-8 Calibration Troubleshooting—Voyager Only Symptom Error displayed when you import a calibration Possible Cause Action .CAL file corrupted Create new .CAL file. See “Exporting .BIC, .MSM, and .CAL files” on page 1-36. Importing a .CAL file generated from a Mariner data file Import a Voyager .CAL. Importing a .CAL file generated from a data file collected in a different instrument mode (Linear, Reflector, or PSD) Import a .
Chapter 9 Troubleshooting 9.5 Printing Troubleshooting Table 9-9 Printing Troubleshooting—Mariner and Voyager Symptom Traces do not print Possible Cause Action Line width is set to 0 or 1 Change the line width. See Section 1.5, Setting Graphic Options. Line color is set to white Change the color. See Section 1.5, Setting Graphic Options.
Peak Detection and Labeling Troubleshooting 9.6 Peak Detection and Labeling Troubleshooting This section includes: • Peak detection and labeling troubleshooting • Charge state and isotope determination troubleshooting Table 9-10 Peak Detection and Labeling Troubleshooting—Mariner and Voyager Symptom Peaks are not detected or labeled Possible Cause Peaks are very close together or label is too long Action • Zoom in on region of interest.
Chapter 9 Troubleshooting Table 9-10 Peak Detection and Labeling Troubleshooting—Mariner and Voyager (Continued) Symptom Peaks are not detected or labeled Possible Cause Analyzing masses above 20,000 Da Increase Mass Resolution setting in Peak Detection Setup. See Section 3.2.2, Strategy for Voyager Peak Detection. %Max Peak Area set too high Decrease. See Section 3.2.2, Strategy for Voyager Peak Detection. %Base Peak Intensity set too high Set to 0.
Peak Detection and Labeling Troubleshooting Table 9-10 Peak Detection and Labeling Troubleshooting—Mariner and Voyager (Continued) Symptom Expected user label not displayed Peak label placed on peak shoulder instead of peak apex Possible Cause Action Delta X value includes more than one peak apex Set Delta X value low enough to prevent the peak labeling windows from overlapping. For information, see Section 3.5.3, Setting Custom Peak Labels.
Chapter 9 Troubleshooting Table 9-11 Peak Detection and Labeling Troubleshooting—Voyager Only Symptom Partially resolved peaks not detected Possible Cause Action Mass resolution set too high to detect average mass Decrease Mass Resolution setting. See Section 3.2.2, Strategy for Voyager Peak Detection. %Base Peak Intensity not adjusted correctly Adjust. See Section 3.2.2, Strategy for Voyager Peak Detection.
Peak Detection and Labeling Troubleshooting Table 9-12 Charge State and Isotope Determination Troubleshooting—Mariner Only (Continued) Symptom Possible Cause Action Known isotope not labeled with charge state Charge State peak labels disabled Turn on Charge State peak labels. See Section 3.5.2, Setting Chromatogram and Spectrum Peak Labels. Max Charge State parameter set too low. See example in “Max Charge State set too low” on page 3-33. Set Max Charge State correctly.
Chapter 9 Troubleshooting Table 9-12 Charge State and Isotope Determination Troubleshooting—Mariner Only (Continued) Symptom Spectrum peaks not labeled with charge state when charge state labels are selected 9 9-20 Applied Biosystems Possible Cause Action Mass of original molecule above 4,000 Da, not range in which the Mariner system can resolve the isotope peaks No action. Normal occurrence. Charge State parameters not set to detect at least two isotope peaks Adjust parameters.
Peak Detection and Labeling Troubleshooting Table 9-12 Charge State and Isotope Determination Troubleshooting—Mariner Only (Continued) Symptom Possible Cause Spectrum peaks labeled with incorrect charge state when charge state labels are selected Charge state determination parameters are set such that peaks are determined to have no charge (continued) Noise between isotope peaks is detected Action Adjust parameters. See: • “Peak Processing parameters (spectrum data only)” on page 3-26 • Section 3.
Chapter 9 Troubleshooting 9 9-22 Applied Biosystems
Appendix A A Warranty Applied Biosystems supplies or recommends certain configurations of computer hardware, software, and peripherals for use with its instrumentation. Applied Biosystems reserves the right to decline support for or impose extra charges for supporting non-standard computer configurations or components that have not been supplied or recommended by Applied Biosystems.
Appendix A Warranty not warrant that the operation of the instrument or software will be uninterrupted or error free. Applied Biosystems will provide any software corrections or “bug-fixes”, if and when they become available, for a period of ninety (90) days after installation.
Warranties limitations THE REMEDIES PROVIDED HEREIN ARE BUYER’S SOLE AND EXCLUSIVE REMEDIES.
Appendix A Warranty A A-4 Applied Biosystems
Appendix B B Overview of Isotopes This appendix contains the following sections: B.1 Isotopes .................................................... B-2 B.2 Monoisotopic and Average Masses ........... B-6 B.3 Isotopes of Common Elements ..................
Appendix B Overview of Isotopes B.1 Isotopes Overview Many elements in their natural state exist as one of several isotopes. An isotope is one of two or more atoms with the same atomic number but a different mass. The most abundant isotope of carbon is 12C, but natural carbon also contains 13C and 14C. Because a mass spectrometer measures mass-to-charge ratios, isotopes appear in the mass spectrum. Isotopes of low abundance, such as 14C, do not affect the appearance of a mass spectrum.
Isotopes As the number of carbon atoms in a compound increases, the possibility of the compound containing a 13C instead of a 12C also increases. A compound with ten carbon atoms includes a molecular ion M+ and an isotopic ion (M+1)+ one mass unit greater than the molecular ion, which is approximately 11 percent of the abundance of the molecular ion. The possibility of including two 13C atoms in the same molecule also increases with increasing number of carbon atoms.
Appendix B Overview of Isotopes M+ (M+1)+ (M+2)+ B Figure B-3 Mass Spectrum of Angiotensin I at Resolution 3,000 In compounds with more than 100 carbon atoms, the height of the first 13C isotope peak exceeds the height of the 12C peak. Isotope-limited resolution The mass range you analyze and the resolving power of the analyzer determine if you observe resolved isotope peaks in a mass spectrum. Figure B-3 represents angiotensin I at a resolution of 3,000.
Isotopes M+ (M+1)+ (M+2)+ B Figure B-4 Mass Spectrum of Angiotensin I at Resolution 1,000 If isotopes cannot be resolved, the highest resolution you can obtain is limited by the width of the isotopic envelope. The isotopic envelope is the mass range of the combined isotopes, as measured at the half height of the tallest isotope peak in the compound (Figure B-5).
Appendix B Overview of Isotopes B.2 Monoisotopic and Average Masses When isotopes are clearly resolved (Figure B-6), the monoisotopic mass is used for mass labeling, and corresponds to the lowest mass peak in the cluster.
Monoisotopic and Average Masses Average mass corresponds to centroid of unresolved peak cluster B Figure B-7 Average Mass Data Explorer™ Software User’s Guide B-7
Appendix B Overview of Isotopes B.3 Isotopes of Common Elements Table B-1 lists the natural abundance of isotopes for some common elements seen in mass spectrometry Table B-1 Isotopes of Common Elements1 B Isotope 1 Mass Natural abundance (%) Isotope Mass Natural abundance (%) H 1.0078 99.985 31 P 30.9737 100 2H 2.0141 0.015 32S 31.9720 95.02 12 12 98.90 33 S 32.9714 0.75 13.0033 1.10 34S 33.9678 4.21 14.0030 99.63 36 35.9670 0.02 15N 15.0001 0.37 35Cl 34.
C Data Explorer Toolbox (Visual Basic Macros) Appendix C This appendix includes: C.1 Overview................................................ C-2 C.2 Preparing Data Before Accessing Macros ....................... C-3 C.3 Accessing the Macros ............................ C-4 C.4 Using the Ladder Sequencing Toolbox .................... C-5 C.5 Using the Peptide Fragmentation Toolbox ............... C-9 C.6 Using the Polymer Analysis Toolbox ..... C-15 C.7 Using MS Fit/MS Tag Toolbox ..........
Appendix C Data Explorer Toolbox (Visual Basic Macros) C.1 Overview Macros provided C The following toolbox of Visual Basic macros is provided with the Data Explorer software: • Ladder SequencingUse when performing sequencing to label peaks with the appropriate amino acid, DNA residue, or RNA residue.
Preparing Data Before Accessing Macros References required The following references are selected by default in the DataExplorerProject in the Visual Basic Editor and are required for the macros in the DataExplorer.VB6 file to successfully run: • • • • • Visual Basic For Applications Data Explorer 4.0 Type Library OLE Automation Microsoft Forms 2.0 Object Library Microsoft Internet Controls To view references: 1.
Appendix C Data Explorer Toolbox (Visual Basic Macros) C.3 Accessing the Macros To access the macros: 1. Open the Data Explorer software. 2. Open a data file. 3. Prepare the data as described in the previous section. 4. From the Tools menu, select Macros. 5. In the Macros dialog box, select modToolBoxPalette.Toolbox_Palette, then click Run. The Toolbox Palette dialog box is displayed.
Using the Ladder Sequencing Toolbox NOTE: If the modToolBoxPalette.Toolbox_Palette is not listed, you must import the macro into the Data Explorer project. For information, see Section 6.7.7, Importing or Exporting Macros in DATAEXPLORER.VB6. Hint: You can assign the modToolBoxPalette.Toolbox_Palette macro to a macro button in the Data Explorer software. For information, see the Data Explorer User’s Guide, Section 6.7, Using the Macro Recorder.
Appendix C Data Explorer Toolbox (Visual Basic Macros) 2. Enter the mass tolerance to apply to the analysis. 3. Click Get Spec Peak List. 4. Remove peaks you do not want included in the calculation by clicking the peak in the list, then clicking Delete Selected Peaks/Adducts. 5. Select the type of spectrum you are examining: Peptide, DNA, or RNA. 6. Under Adducts to Remove, select the items you do not want included in the interpretation.
Using the Ladder Sequencing Toolbox 7. 8. Under Annotate Spectrum, select the types of labels you want displayed: • Reference Mass (*)Mass of the reference peak against which the current peak is compared. • Mass DifferenceDifference between the current peak and the reference, preceded by a minus sign (–). • Peak MassMass of the current peak, preceded by an equal sign (=). Click Label Peaks.
Appendix C Data Explorer Toolbox (Visual Basic Macros) If DNA or RNA is selected, the software: Displaying the original labels C C-8 Applied Biosystems • Examines the spectrum in 270 Da increments and selects the most intense ion in the range. (The 270 Da increment is used because it is less than the smallest mass difference related to a DNA or RNA base.) • Labels mass differences (plus or minus the specified Tolerance) that correspond to DNA (ACGT) or RNA (ACGU) bases.
Using the Peptide Fragmentation Toolbox C.5 Using the Peptide Fragmentation Toolbox Use the Peptide Fragmentation toolbox when examining Voyager composite PSD spectra or Mariner in-source CID spectra to label immonium ions, identify fragment ion pairs, view sequences based on different reference peaks, and determine if a selected peak is of a specific fragment ion category. Setup To enter Setup parameters for the Peptide Fragmentation Toolbox macro: 1.
Appendix C Data Explorer Toolbox (Visual Basic Macros) 3. Add peaks to the peak list to be included in the calculation by typing a mass in the Add Mass to Peak List field, then clicking Add Peak. Remove unwanted peaks from the list that you do not want included in the calculation by clicking the peak in the list, then clicking Delete Selected Peaks. 4. Pairs If you will be identifying y and b pairs, select the precursor peak, then click Use Selected Peak. To list ion pairs: 1. Click the Pairs tab.
Using the Peptide Fragmentation Toolbox Sequence • y and b pairsLists peak pairs whose combined masses plus 1 Da add up to the Precursor Ion Mass you specified on the Setup tab. • Loss of H2O Lists peak pairs with a 18 Da mass difference. 3. To remove all pairs results, click Clear List. 4. To remove a mass from the Spec Peak List (to simplify the sequence interpretation), select an entry in the pairs results, then click Left or Right under Remove Mass. 5.
Appendix C Data Explorer Toolbox (Visual Basic Macros) 2. If desired, click Label Immonium Ions. NOTE: Label immonium ions before selecting a reference peak and starting the search. If you click Label Immonium Ions after selecting a reference peak, the amino acid labels applied to the spectrum are erased, and mass labels are reapplied. 3. Select a peak in the Spec Peak List, then click Set Selected Peak as Reference.
Using the Peptide Fragmentation Toolbox 5. Correlation Click Copy to Output Window Result Tab to copy results to the Result tab. You can then copy from the Result tab to another application such as Notepad, or print, as needed. To list ion pair correlations: 1. Click the Correlation tab. 2. Select a peak in the Spec Peak List. 3.
Appendix C Data Explorer Toolbox (Visual Basic Macros) 4. Click Find Correlation. Correlations for the selected peak are listed. NOTE: Yp, Sp and Tp represent phosphotyrosine, phosphoserine, and phosphothreonine, respectively. Displaying the original labels C C-14 Applied Biosystems 5. Click Copy to Output Window Result Tab to copy results to the Result tab. You can then copy from the Result tab to another application such as Notepad, as needed. 6.
Using the Polymer Analysis Toolbox C.6 Using the Polymer Analysis Toolbox Use the Polymer Analysis Toolbox to determine the following values that define the molecular distribution of a polymer: • • • • Mn—Number average molecular weight Mw—Weight average molecular weight Mz—z-average molecular weight Mw/Mn—Polydispersity Index The Polydispersity Index represents how widely dispersed the polymeric distribution is. A lower value (for example, 1.02) indicates a narrowly dispersed polymer.
Appendix C Data Explorer Toolbox (Visual Basic Macros) 3. Select the mode for the analysis: • Use the entire mass range—Calculates average molecular weights using all peak intensities within the X Display Range. It does not distinguish between different polymeric species that may be present in the mass range. Enter an adduct mass. C Figure 3-14 Polymer Analysis Toolbox NOTE: If the predominant ion series is M+Na+, enter 23 for the adduct ion.
Using the Polymer Analysis Toolbox • Use labeled peaks—Calculates average molecular weights using areas of peaks listed in the Spec Peak List. It allows calculation of values for a distinct polymer series when two or more species are present. Enter an adduct mass. Enter the mass of the end group to subtract from the calculation. You can leave this field blank if you do not know the end group mass. CAUTION: Correct labeling of the peaks is essential when using the labeled peaks option. 4.
Appendix C Data Explorer Toolbox (Visual Basic Macros) C.7 Using MS Fit/MS Tag Toolbox Use the MS Fit/MS Tag toolbox when analyzing protein digest, peptide, or peptide fragment spectra to perform a protein database search. Preparing data before accessing C Running MS Fit/MS tag Before using the MS Fit/MS Tag toolbox: 1. In the Data Explorer software, smooth the data, perform a baseline correction, then deisotope. 2.
Using MS Fit/MS Tag Toolbox 2. Click: • MS-Fit tabIf you are examining peptide data from a protein digest. • MS-Tag tabIf you are examining PSD data. 3. Navigate to the web site containing the database to search. 4. Adjust settings on the web site as needed. 5. Minimize the web page. Do not exit. 6. In the MS Tag dialog box, enter Parent Ion mass if you are using MS-Tag. 7. Click Copy Peaks. The Spec Peak list for the data file is copied to the web page. 8.
Appendix C Data Explorer Toolbox (Visual Basic Macros) C C-20 Applied Biosystems
Index Numerics and Symbols - in spectrum header 2-31 %Base Peak Intensity definition, chromatogram 3-20 definition, spectrum 3-22 in peak detection algorithm 3-67 Mariner data 3-7 setting for active detection range, spectrum 3-30 setting global, spectrum 3-22 setting with data cursor, chromatogram 3-11, 3-16 setting with data cursor, spectrum 3-23 setting, chromatogram 3-20 Voyager data 3-10 %Max Peak Area definition, chromatogram 3-20 definition, spectrum 3-23 Mariner data 3-7 setting for active detection
I N D E X Amino acids, labeling C-5 Analog signal, displaying 4-2 Analyzer Temperature, displaying trace 4-2 ANGIOTENSIN_FRAGMENTS.
Bar mode, traces 1-28 BAS files for macros 6-43 Base mass, labeling peaks 3-55, 3-56 Base peak intensity definition 4-2 scaling to 2-12 threshold for peak detection, chromatogram 3-20 threshold for peak detection, spectrum 3-22, 3-30 Baseline changing line width 1-26 displaying on trace 3-55, 3-58 Baseline correction chromatogram 4-29 spectrum 5-47 spectrum, advanced 5-48 Baseline offset chromatogram 4-27 spectrum 5-45 Basic peak detection parameters description, chromatogram 3-19 description, spectrum 3-22
I N D E X Calibrating mass, automatic (Mariner data only) (continued) reverting to instrument calibration 5-22 troubleshooting 9-9 turning on 5-34 when to use 5-28 Calibrating mass, manual see also Calibration constants baseline correcting and deisotoping to optimize mass accuracy 7-14 calibration reference file (REF) 5-17 commands dimmed on menu 9-11 constants, applying A and B to new file 5-16 error, fit 5-12 error, initial 5-12 fit outliers, eliminating 5-12 importing, Voyager requirements 5-25 multi-po
Calibration constants see also Calibrating mass applying to new file 5-16, 8-20 calculating 5-34 displayed in Output window 5-14, 8-18 exporting 1-36 extracting from DAT file 1-36 importing from another source 5-16, 8-20 PSD 8-20 reverting to original instrument 5-22 Calibration curve, peak weighting factors 5-10, 8-14 Calibration fit error 5-12 Calibration reference file (REF) ANGIOTENSIN_FRAGMENTS.
I N D E X Charge state, peak (continued) requirements for labeling 3-53 single 5-59 tolerance calculation 3-32 troubleshooting 9-17 z labels 3-58 zero 3-40, 3-43 Chro peak list tab, Output window 1-15 CHRO window, see Chromatogram window Chromatogram noise threshold calculated automatically 3-21, 3-68 in peak detection algorithm 3-68 Chromatogram window see also Chromatogram window, traces see also Peak Labels see also Traces adding and subtracting spectra 4-20 baseline correction 4-29 baseline offset 4-27
CombiSolv data displaying one injection 4-24 Event Tag Filtering command dimmed (Mariner data only) 4-24 Comment acquisition, displaying 1-15 acquisition, displaying for open data file 1-15 acquisition, displaying when opening a data file 2-3 result file 1-15, 2-40 Comparing data files 2-38 Composite spectrum, PSD automatically processed when generated 8-6 displaying segment traces 8-3 generating 8-2, 8-5 how it is generated 8-6 labeling fragment ions 8-8 Max Stitch Masses 8-4 precursor mass, changing 8-23
I N D E X Customizing (continued) SET files 1-17 toolbars 1-22 D DAD displaying Channels 1-12, 4-2 displaying TAC 1-12 displaying traces 2-6 extracted absorbance chromatogram (XAC) 4-13 in spectrum header 2-32 spectrum, displaying 4-2, 5-2 TAC in chromatogram header 2-30 XAC in chromatogram header 2-30 Dark background changing colors 1-20 default settings 1-23 SET file 1-19 DAT format converting DAT file to ASCII 1-34 converting from SPC 1-30 converting profile to centroid 1-33 extracting information from
Data file (continued) full name not displayed 1-14 inserting traces into 2-37 moving between open 2-8 multiple, zooming 2-36 name 1-14 names do not print 9-13 opening 1-30 opening and applying default and selected settings 2-4 opening and running a macro automatically 6-45 opening manually 2-2 opening PSD 8-2 read-only 2-7 result, opening 2-39, 2-40 switching between open 2-8 trace browser 2-8 version of software used to acquire 1-15 working with separate 2-36 zooming multiple 2-36 Data format, see DAT form
I N D E X Detection Ranges (continued) setting manually, chromatogram 3-19, 3-20 setting manually, spectrum 3-28 setting parameters globally, spectrum 3-22 setting parameters locally, spectrum 3-30 Detection, see Peak detection DI in spectrum header 2-32, 3-49 Diode array detector data, see DAD Diode array detector data, see DAD data Disk space, conserving by converting profile data to centroid 1-33 Display range scaling 2-11 x range, expanding 2-11 y range, expanding 2-11 Display Trace dialog box 2-18 Dis
Excel, see Microsoft Excel Exiting software 1-3 Expanding traces 2-21 Exporting see also Converting ASCII data 1-34 BIC 1-36 CAL files 1-36 Configuration from DAT file 1-36 entire data file 1-34 macros from DATA EXPLORER.
I N D E X Filter Width Increment setting manually, spectrum 3-31 suggested value, spectrum 3-31 value used when resolution-based peak detection enabled 3-31 Filtered traces, viewing, see Filtering Filtering chromatogram traces 4-23 event tags (Mariner data only) 4-24 monoisotopic peaks 3-43 noise, chromatogram 4-17 peak list 3-42 Fit Error, calibration 5-12 Formulas determining if present in observed spectrum 6-31 determining possible for a mass 6-2 Fragment ions correspond to loss of 4-9 determining if ma
I Immonium ions, labeling C-9 IMMONIUM_IONS.REF 5-18, 8-19 Import Calibration error displayed 9-11, 9-12 procedure 5-16 PSD 8-20 Importing macros into DATA EXPLORER.
I N D E X Isotope Match Intensity, elemental targeting 6-33 Isotope Match Score elemental composition 6-6 elemental composition, not reported for fragment ion calculations 6-6 elemental targeting 6-33 K Keywords, Windows NT entering 1-31 searching 1-32 viewing 1-32 L Labeling peaks see also Peak labels chromatogram 3-54 customizing label appearance 1-25 factors affecting 3-52 manually 3-39 monoisotopic 3-43 partially resolved 7-11 spectrum 3-56 with amino acid, DNA, or RNA labels C-5 with area 3-58 with
M Macro Recorder advanced editing 6-42 buttons, assigning to macros 6-38 DATAEXPLORER.VB6 location 6-35 DATAEXPLORER.VB6 not overwritten when new software installed 6-43 deleting a macro 6-41 description 6-34 exporting macros from DATA EXPLORER.VB6 6-44 functions not supported 6-35 importing macros into DATA EXPLORER.
I N D E X Mass Calibration commands dimmed on menu 9-11 not displayed on menu 9-8 Mass calibration, see Calibrating mass Mass, centroid calculating 3-39 copying from peak list 1-41 definition B-6 displaying peaks as histograms 5-36 labeling 3-57 Mass deconvolution command dimmed on Process menu 5-37, 9-8 example 7-4 performing 5-37 result, failed to calculate 9-8 results 5-40 Mass Difference peak label from adjacent peak (regular labels) 3-57 from adjacent peak (user labels) 3-62 from selected peak 3-57 Ma
MS Method (Mariner data only) (continued) instrument settings, viewing 4-23 spectrum numbers in filtered trace 4-25 MSM files extracting from DAT file 1-36 overview 1-7 Multiple Charge command dimmed on menu 5-37, 9-8 not displayed on menu 9-8 Multiple data files comparing 2-36, 2-38 copying traces into a window 2-37 printing 2-36 working with separately 2-36 zooming 2-13, 2-36 Multiple spectra in Voyager data files 2-7 Multiply charged peaks deconvoluting 5-37 single-charge conversion 5-59 Multi-point cali
I N D E X Output window acquisition comment, displaying 1-15 calibration statistics, displaying 5-13, 8-18 Chro Peak list tab 1-15 clearing 1-16 closing 1-16 copying results from 2-28 instrument settings 1-16 Instrument Settings tab 1-15 peak list, chro 1-15 peak list, displaying 1-15 peak list, importing and saving in Excel 3-41 peak list, saving as a file 3-40 peak list, spec 1-15 Results tab 1-15 results, displaying 1-15 sample info 1-15 Sample Info tab 1-15 Spec Peak list tab 1-15 Overlapping peak labe
Peak detection (continued) Noise Threshold, calculated automatically for chromatogram data 3-21, 3-68 overview 3-2 Peak Processing parameters, spectrum 3-16, 3-26 peak start and end, displaying 3-55, 3-58 peaks do not appear in spectrum 9-17 process that occurs during 3-67 proteins 3-6 ranges, overlapping 3-5 regions, setting chromatogram 3-19, 3-20 regions, setting spectrum 3-28 resetting Basic settings 3-18 spectrum 3-68 troubleshooting 9-14 use same settings for all traces 3-21, 3-25 Peak detection param
I N D E X Peak labels (continued) centroid 3-56 chromatogram, setting 3-54 custom 3-61 custom, creating for fragment spectra 8-9 customizing 1-25 decimal places displayed 3-55, 3-56 deleting from trace 3-44, 3-59 displaying 3-65 DNA C-5 extracting from DAT file 3-64 factors affecting 3-52 height 3-55, 3-56 horizontal 3-55, 3-58 immonium ions C-9 manually applying 3-39, 3-52 manually inserting peaks 3-39 monoisotopic 3-43 not displayed 3-59, 3-66, 9-14, 9-15 overlapping 3-55, 3-58 peak start and end, displa
Peak weighting factors 5-10, 8-14 Peak Width minimum and maximum used 3-21, 3-25 set automatically by software 3-21, 3-25 Peaks, do not appear in spectrum 9-17 Peptide fragmentation macro C-2, C-9 Periodic table 6-10 PerSeptive Biosystems Technical Support, see Applied Biosystems Technical Support PKT files 3-40 Points across a peak, determining 3-21, 3-31, 5-51 Polydispersity index, determining C-15 Polymer analysis macro C-2, C-15 Positive ion z label 3-58 Precursor mass, see PSD analysis Preface xi Previ
I N D E X PSD analysis (continued) composite spectrum, displaying 8-2, 8-5 composite spectrum, how it is generated 8-6 fragment labels, applying 8-8 optimum resolution observed near Max Stitch Mass 8-4 peak detection algorithm 3-5 peak detection, checking 8-10 Peptide fragmentation macro C-9 precursor mass, changing 8-23 PSD, overview of creating 8-10 REF file, creating 8-21 segment labels, applying 8-8 segment traces, displaying 8-3 PSD calibration equation 8-6 Precursor mass, impact of changing 8-23 PSD
Result tab, Output window 1-15 Results see also RSD and RCD annotating traces with 2-28 copying 2-28 displaying in Output window 1-15 elemental composition 6-5 elemental targeting 6-33 exporting 2-39 extracting information from 1-36 ion fragmentation 6-29 isotope 6-19 mass deconvolution 5-40 name of raw data file result is derived from 1-15 peak list 3-38 RCD and RSD files 2-39 resolution, mass 6-22 signal-to-noise ratio 6-24 Results (DAT) copying 2-28 deleting results 2-38 opening results 2-38 saving resul
I N D E X Sequence Control Panel, Mariner, automatic calibration settings (reference masses) for 5-27 Sequence Control Panel, Voyager, automatic calibration settings (reference masses) for 5-28 SET files applying 1-20 applying when opening data file 2-4 contents 1-18 creating for Mariner Sequence Control Panel 5-27 creating for Voyager Sequence Control Panel 5-28, 5-29 customizing 1-19 defaults 1-19, 1-23 description 1-7 extracting from DAT 1-37 modifying 1-19 restoring 1-20 saving for use with other data
Spectra (continued) summing non-contiguous 4-21 troubleshooting 9-17 truncating 5-56 types of 5-2 Spectrum noise threshold, setting locally 3-30 Spectrum window see also Peak labels see also Spectrum window, traces see also Traces adding spectra from different data files 5-64 adding spectra from same data file 5-4 baseline correction 5-47 baseline correction, advanced 5-48 baseline offset 5-45 centroid, creating 5-36 centroiding 3-69, 5-36 charge state labels incorrect 3-60, 9-20 charge state labels not dis
I N D E X T Tabs for open files 2-8 in Data Explorer window 2-8 TAC in chromatogram header 2-30 Mariner data, optional 1-12 Tag, see Event tag Target compounds determining if present in spectrum 6-31 Technical support contacting 9-2 for computers with altered configuration A-1 Temperature trace, displaying 4-2 Text files saving peak list in PKT 3-40 Threshold, peak detection chromatogram 3-20 global, spectrum 3-22 local, spectrum 3-30 TIC see also Chromatogram window description 4-2, 4-3 Title when saving
Traces (continued) Replace mode, setting 2-17 scaling mode, setting 2-12 splitting 2-15 switching between 2-8 text, customizing 1-25 traces do not print 1-26, 2-33 type, selecting in Chromatogram window 4-2 use same graphic options settings for all 1-24 UV, offset 4-30 vertical bar mode 1-28 white, does not print 1-26, 2-33 Traces, copying from different data file 2-37 to Windows clipboard 1-38 to WMF 1-39 Traces, removing active 2-21 from history list 2-22 inactive 2-21 Troubleshooting calibration 9-9 cali
I N D E X V Valley-to-Baseline integration chromatogram 3-21, 3-70 spectrum 3-26, 3-70 Valley-to-Valley integration chromatogram 3-21, 3-70 spectrum 3-26, 3-70 Version of software used to acquire data 1-15 Vertical bars displaying centroid traces 5-36 traces do not print 2-34 Vertical cursor 1-27 Vertical peak labels 3-55, 3-58 Vial number, displaying in Chromatogram window 3-55 Viewing read-only files 2-7 Views, linking 2-13 Visual Basic Editor 6-42 Visual Basic macros provided see also Data Explorer Tool
X I N D E X X cursors, setting 1-27 x,y data pairs, copying 1-39 XAC in chromatogram header 2-30 see also Extracted absorbance chromatogram (XAC) X-axis chromatogram 2-11 setting range 2-11 spectrum 2-11 XIC see also Extracted ion chromatogram (XIC) in chromatogram header 2-31 Y y and b ion pairs, labeling C-11 Y cursor, setting 1-27 Y-axis offsetting chromatogram 4-27 offsetting spectrum 5-45 scaling 2-12 spectrum 2-11 Z z labels 3-58 z-average molecular weight, determining C-15 Zero charge state displ
I N D E X Index-30 Applied Biosystems
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