Applied Biosystems Data Explorer 4 Series User Manual
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Applied Biosystems Data Explorer 4 Series User Manual

Applied Biosystems Data Explorer 4 Series User Manual

Data explorer user guide v4 series sw user guide
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Summary of Contents for Applied Biosystems Data Explorer 4 Series

  • Page 1: User Guide

    Data Explorer ™ Software Version 4 Series Software User Guide...
  • Page 2 AB (Design), Applera, Biospectrometry, CombiSolv, Data Explorer, Mariner, and Voyager are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. HP and Laserjet are registered trademarks of Hewlett-Packard Co. Microsoft, PowerPoint, Visual Basic, and Windows NT are registered trademarks of Microsoft Corporation.

  • Page 3: Table Of Contents

    Table of Contents How to Use This Guide Chapter 1 Data Explorer™ Basics Overview File Formats and Types 1.2.1 Software Applications Compatibility ... 1-5 1.2.2 Data (.DAT) File Format ... 1-5 Parts of the Data Explorer Window Customizing the Data Explorer Window 1.4.1 Setting Default Values ...1-17 1.4.2...
  • Page 4 Chapter 2 Using Chromatogram and Spectrum Windows Opening and Closing Data Files 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 2.1.5 Moving Between Open Files ... 2-8 2.1.6 Closing Data Files ...2-10 Adjusting the Display Range...
  • Page 5 Chapter 3 Peak Detection and Labeling Overview 3.1.1 Default Peak Detection ... 3-2 3.1.2 The Resolution-Based Peak Detection Routine ... 3-3 Peak Detection 3.2.1 Strategy for Mariner Peak Detection ... 3-6 3.2.2 Strategy for Voyager Peak Detection ... 3-8 3.2.3 Setting Peak Detection Parameters ...
  • Page 6 Chapter 4 Examining Chromatogram Data Overview Creating an Extracted Ion Chromatogram 4.2.1 Creating an Extracted Ion Chromatogram (XIC) ... 4-5 4.2.2 Creating a Constant Neutral Loss (CNL) Chromatogram ... 4-9 Creating an Extracted Absorbance Chromatogram (XAC) (Mariner Data Only) Noise Filtering/Smoothing Adding and Subtracting Raw Spectra Within a Data File Displaying MS Method Data (Mariner Data Only) Adjusting the Baseline...
  • Page 7 5.12 Adding and Subtracting Raw or Processed Spectra from the Same or Different Data Files (Dual Spectral Trace Arithmetic) Chapter 6 Using Tools and Applications Using the Elemental Composition Calculator 6.1.1 Determining Elemental Composition ... 6-2 6.1.2 Setting Limits ... 6-7...
  • Page 8 Chapter 7 Data Explorer Examples Mariner Data Examples 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 7.2.1 Detecting and Labeling Partially Resolved Peaks ... 7-11 7.2.2 Processing Before Calibrating to Optimize Mass Accuracy ...7-14 7.2.3...
  • Page 9 Appendix A Warranty Information Appendix B Overview of Isotopes Appendix C Data Explorer Toolbox (Visual Basic Macros) Overview Preparing Data Before Accessing Macros Accessing the Macros Using the Ladder Sequencing Toolbox Using the Peptide Fragmentation Toolbox Using the Polymer Analysis Toolbox Using MS Fit/MS Tag Toolbox Index ™...
  • Page 10 Table of Contents Applied Biosystems...

  • Page 11: How To Use This Guide

    How to Use This Guide Purpose of this guide Audience Structure of this guide Chapter/Appendix Chapter 1, Data Explorer™ Basics Chapter 2, Using Chromatogram and Spectrum Windows Chapter 3, Peak Detection and Labeling Chapter 4, Examining Chromatogram Data The Applied Biosystems Data Explorer Guide describes processing and analyzing data with the Data Explorer software.
  • Page 12 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.
  • Page 13 Conventions Notes, Cautions, Warnings, and Hints 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.

  • Page 14: Related Documentation

    Related documentation Send us your comments Applied Biosystems 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.

  • Page 15: Data Explorer ™ Basics

    Chapter ™ 1 Data Explorer Basics 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 ...

  • Page 16: Chapter 1 Data Explorer™ Basics

    Chapter 1 Data Explorer™ Basics 1.1 Overview Description Features Applied Biosystems ™ The Data Explorer Version 4.0 processing software is graphical software that you use to analyze, calibrate, and report data. You can use the Data Explorer software to analyze data collected on: ™...

  • Page 17: Exiting The Software

    Overview Starting and To start the Data Explorer software from the Windows NT desktop, double-click the Data Explorer icon on the desktop. exiting the The Data Explorer window opens. software The Data Explorer window is blank with only a few menus displayed until you open a data file.
  • Page 18 Chapter 1 Data Explorer™ Basics Figure 1-2 Data Explorer Window with Voyager Data Default colors Applied Biosystems 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.

  • Page 19: File Formats And Types

    1.2 File Formats and Types This section describes: 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.
  • Page 20 Chapter 1 Data Explorer™ Basics Mariner .SPC file format Voyager .MS file format Extracting information from .DAT files Table 1-1 Information Stored In a .DAT File Category File Type Settings .BIC Applied Biosystems In Mariner software versions earlier than version 3.0, data files are stored in .SPC format.
  • Page 21 Table 1-1 Information Stored In a .DAT File (Continued) Category File Type Settings .MSM MS Method settings, if data was acquired using an (Mariner .MSM file. (continued) only) NOTE: To access the instrument settings used to acquire each spectrum in an MS Method, you must first extract the .MSM file from the data file, then export the .BIC files from the .MSM file using the Export button in the MS Method editor.

  • Page 22: Additional Files

    Chapter 1 Data Explorer™ Basics Additional files types Category File Type Data .PKT .TXT Data .SPC (Mariner only) (Voyager only) .MSA and .MSF (Voyager only) .MSB (Voyager only) Procedure .TUN (Mariner only) Applied Biosystems Additional file types you may see on your system are described below.
  • Page 23 Table 1-2 Additional File Types (Continued) Category File Type Reference .REF List of masses to select from during calibration. See “Creating and saving a calibration reference file” on page 5-18. Process .RCT Results file saved from: (Mariner only) NOTE: Results for a .DAT file are stored within the .DAT file, not as a separate file.
  • Page 24 Chapter 1 Data Explorer™ Basics Table 1-2 Additional File Types (Continued) Category File Type Process .RCD (continued) .RSD 1-10 Applied Biosystems File Content Chromatogram results file exported from .DAT files. See Section 2.7, Exporting, Opening, and Deleting .RCD and .RSD Results Files (Mariner Data Only). Spectrum results file exported from .DAT files.

  • Page 25: Parts Of The Data Explorer Window

    1.3 Parts of the Data Explorer Window This section describes: Overview Figure 1-3 shows the Data Explorer window with Mariner data. Toolbar Chromatogram window Data file name Spectrum window Tabs for open data files Output window Figure 1-3 Parts of the Data Explorer Window Parts of the Data Explorer Window •...
  • Page 26 Chapter 1 Data Explorer™ Basics Toolbar Chromatogram and Spectrum windows Table 1-3 Mariner Data Displayed in Chromatogram and Spectrum Windows Window CHRO Displays: • Total Ion Chromatogram—Includes the entire mass range saved in the data file. • Extracted Ion Chromatogram (XIC) (optional)—Includes only the signal response from a mass window or range •...
  • Page 27 Table 1-3 Mariner Data Displayed in Chromatogram and Spectrum Windows Window SPEC 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.

  • Page 28: Context-Sensitive Menus

    Chapter 1 Data Explorer™ Basics Context-sensitive menus Tabs for data files Data file names 1-14 Applied Biosystems 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. For more information on Chromatogram and Spectrum windows, see Chapter 2, Using Chromatogram and Spectrum Windows.

  • Page 29: Output Window

    • Section 5.3, Manual Calibration, or Section 5.4, Automatic Calibration • Section 5.6, Mass Deconvolution (Mariner Data Only) • Section 6.2, Using the Isotope Calculator • Section 6.3, Using the Mass Resolution Calculator • Section 6.4, Using the Signal-to-Noise Ratio Calculator Chro Peak List —Displays results of chromatogram...
  • Page 30 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 6.1, Using the Elemental Composition Calculator. • Elemental Targeting—Displays results for the Elemental Targeting application.

  • Page 31: Customizing The Data Explorer Window

    1.4 Customizing the Data Explorer Window This section includes: 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.
  • Page 32 Chapter 1 Data Explorer™ Basics Overview of processing and graphic settings What settings contain Default processing and graphic settings 1-18 Applied Biosystems 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.
  • Page 33 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.
  • Page 34 Chapter 1 Data Explorer™ Basics Opening, customizing, and saving .SET files Applying a .SET file 1-20 Applied Biosystems To open, customize, and save .SET files: 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”...

  • Page 35: Customizing Toolbars

    To use the Settings option: 2. Select one of the following: 3. If you select a Restore Settings option, select or type the Hint: You can also restore default processing and graphic settings when you open a file or files. See Section 2.1, Opening and Closing Data Files.
  • Page 36 Chapter 1 Data Explorer™ Basics Undocking toolbars Creating toolbars 1-22 Applied Biosystems 4. 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.

  • Page 37: Setting Graphic Options

    1.5 Setting Graphic Options This section includes: NOTE: Changes you make to Graphic Options are saved with the data file. 1.5.1 Changing Background Color White or dark You can switch background color by selecting Default from the Display menu, then selecting: background NOTE: These .SET files contain graphic settings only.

  • Page 38: Customizing Graphic Options

    Chapter 1 Data Explorer™ Basics 1.5.2 Customizing Graphic Options Accessing graphic options 1-24 Applied Biosystems This section includes: • Accessing graphic options • Setting colors • Setting line widths • Setting data cursors • Setting traces in line or bar mode •...

  • Page 39: Setting Colors

    Line or vertical bar traces Line width Data cursor Graphic compression Figure 1-4 Graphic Options Dialog Box— Graph Setup Tab Setting colors You can set colors manually or automatically. Manually To manually select the color of graph features (axis, peak bounds, tick labels, data cursor) and plot features (traces, peak labels): Select Graphic Options from the Display menu.
  • Page 40 Chapter 1 Data Explorer™ Basics Automatically using Auto Color Setting line widths 1-26 Applied Biosystems When you manually set colors, note: • 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.

  • Page 41: Setting Data

    Setting data To enable data cursors and set cursor labels and attributes: cursors Select Graphic Options from the Display menu. 2. In the Data Cursor section of the Graph Setup tab (see Figure 1-4 on page 1-25), select Show Data Cursors, then select one of the following cursors from the Type drop-down list: •...
  • Page 42 Chapter 1 Data Explorer™ Basics Setting traces in Line or Vertical Bar mode Setting graphic compression 1-28 Applied Biosystems 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.

  • Page 43: Reverting To Previous Graphic Options

    1.5.3 Reverting to Previous Graphic Options You have two options to revert to previously used graphic options: Hint: Instead of applying the settings saved with a data file, you can apply the default settings stored in the default .SET file for your system (see page 1-18). To apply the default settings, close the data file, open the data file again, then select Use Default Settings in the Open dialog box.

  • Page 44: Extracting And Saving Information

    Chapter 1 Data Explorer™ Basics 1.6 Managing Files 1.6.1 Converting .SPC File Format to .DAT File Format (Mariner Data Only) When to convert 1-30 Applied Biosystems This section describes: • Converting .SPC file format to .DAT file format (Mariner only) •...

  • Page 45: Before You Begin

    Before you begin Confirm that the .SPC and .CGM files are located in the same directory. Use Windows NT 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”...

  • Page 46: Viewing File Properties

    Chapter 1 Data Explorer™ Basics Viewing file properties Searching file properties 1-32 Applied Biosystems The Convert to .DAT Format dialog box reappears. 5. Click OK. A message box is displayed, showing the file name of the newly created .DAT file. 6.

  • Page 47: Converting Data From Profile To Centroid (Mariner Data Only)

    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.

  • Page 48: Converting To And Exporting Ascii Data

    Chapter 1 Data Explorer™ Basics 1.6.3 Converting to and Exporting ASCII Data Converting a data file to ASCII format Exporting a trace to ASCII format 1-34 Applied Biosystems This section describes: • 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...

  • Page 49: Importing A Trace In Ascii Format

    1.6.4 Importing a Trace in ASCII Format 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.

  • Page 50: Extracting And Saving Information From .Dat, .Rsd, And .Rcd Files

    Chapter 1 Data Explorer™ Basics 1.6.5 Extracting and Saving Information from .DAT, .RSD, and .RCD Files Overview Exporting .BIC, .MSM, and .CAL files 1-36 Applied Biosystems 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.
  • Page 51 3. In the Save As dialog box, type a name for the exported Saving .SET files To save processing and graphic settings (.SET) from a .DAT or results file: 2. From the File menu, select Settings, then select one of 3.

  • Page 52: Copying From Data Files

    Chapter 1 Data Explorer™ Basics Saving .LBS and .LBC files 1.6.6 Copying from Data Files Overview Copy trace image 1-38 Applied Biosystems 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.
  • Page 53 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: Select the trace window to copy.
  • Page 54 Chapter 1 Data Explorer™ Basics Copy all peaks 1-40 Applied Biosystems 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.
  • Page 55 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.
  • Page 56 Chapter 1 Data Explorer™ Basics 1-42 Applied Biosystems...
  • Page 57 Using Chromatogram and Spectrum Windows This chapter contains the following sections: Opening and Closing Data Files ... 2-2 Adjusting the Display Range ... 2-11 Organizing Windows ... 2-13 Manipulating Traces ... 2-14 Working with Multiple Data Files ... 2-36 Saving, Opening, and Deleting .DAT Results ... 2-38 Exporting, Opening, and Deleting .RCD and .RSD Results Files (Mariner Data Only) ...

  • Page 58: Chapter 2 Using Chromatogram And Spectrum Windows

    Chapter 2 Using Chromatogram and Spectrum Windows 2.1 Opening and Closing Data Files 2.1.1 Opening Data Files Opening a recently opened file Opening data files with File Open Applied Biosystems This section includes: • Opening data files • Displaying Mariner UV traces •...
  • Page 59 Figure 2-1 Select Files Dialog Box Click the down arrow to display the Files of type list, then select the file extension to display. 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.

  • Page 60: Specifying Settings

    Chapter 2 Using Chromatogram and Spectrum Windows Specifying settings Applied Biosystems 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): •...
  • Page 61 Opening and Closing Data Files Figure 2-2 Data Explorer Window with Four Mariner Data Files Open (Each .DAT File Displays a Chromatogram and a Spectrum Trace) Figure 2-3 Data Explorer Window with Four Voyager Data Files Open (Spectrum Traces Only Displayed by Default) ™...

  • Page 62: Chapter 2 Using Chromatogram And Spectrum Windows

    Chapter 2 Using Chromatogram and Spectrum Windows Type of Data Mariner data 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.

  • Page 63: Displaying Voyager Chromatograms

    2.1.3 Displaying Voyager Chromatograms To display chromatograms for multispectrum Voyager .DAT files: 2. To display a Chromatogram window, click a Spectrum 2.1.4 Viewing Read-Only Files For quick scanning of archived data files, you can view read-only files using the File Open dialog box. When you open a read-only file, a message indicates that the file is read-only and prompts you to open the file.

  • Page 64: Moving Between Open Files

    Chapter 2 Using Chromatogram and Spectrum Windows 2.1.5 Moving Between Open Files Using tabs Click a tab to select an open data file Using Activate File Applied Biosystems You can have more than one file open at a time. You can move between open files in three ways: •...
  • Page 65 Figure 2-5 Select File to Activate Dialog Box 3. Select: • Maximize—To maximize the Spectrum window of the selected file • Activate—To activate the Spectrum window of the selected file Data Explorer Opening and Closing Data Files ™ Software User’s Guide...

  • Page 66: Closing Data Files

    Chapter 2 Using Chromatogram and Spectrum Windows 2.1.6 Closing Data Files 2-10 Applied Biosystems 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.

  • Page 67: Adjusting The Display Range

    2.2 Adjusting the Display Range To set the display range: 2. From the Display menu, select Range. 3. Select X Range to set the x-axis range. The scaling units 4. Select Y Range to scale the y-axis range. Click the Chromatogram or Spectrum window to activate it.
  • Page 68 Chapter 2 Using Chromatogram and Spectrum Windows Parameter Scaling Mode Display Relative Autoscales the trace to the largest peak in the selected range. Base Peak Autoscales the trace to the base peak in the entire range, not the Relative selected range. Displays a right-axis label with the base peak intensity.

  • Page 69: 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.

  • Page 70: Manipulating Traces

    Chapter 2 Using Chromatogram and Spectrum Windows 2.4 Manipulating Traces 2.4.1 Zooming, Centering, and Customizing a Trace Zooming and unzooming 2-14 Applied Biosystems This section includes: • Zooming centering, and customizing a trace • Duplicating a trace • Dividing the active trace •...

  • Page 71: Duplicating A Trace

    Centering a peak To center a peak in the trace window: in the trace 2. Click the Spec Peak List or Chro Peak List tab in the 3. Double-click the peak to center. Customizing a See Section 1.5, Setting Graphic Options, for information on customizing trace parameters such as line widths and data trace compression.

  • Page 72: Adding Traces From The Same Data File To A Window

    Chapter 2 Using Chromatogram and Spectrum Windows 2.4.4 Adding Traces from the Same Data File to a Window Overview 2-16 Applied Biosystems 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: •...
  • Page 73 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.
  • Page 74 Chapter 2 Using Chromatogram and Spectrum Windows Adding a trace 2-18 Applied Biosystems 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.
  • Page 75 Manipulating Traces Original trace 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).
  • Page 76 Chapter 2 Using Chromatogram and Spectrum Windows Original trace Added traces Figure 2-9 Added Traces Containing Data 2-20 Applied Biosystems 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). For a description of trace labels, see “Viewing Trace Labels”...

  • Page 77: Removing Traces

    2.4.5 Removing Traces Removing the To remove the active trace from a window: active trace 2. Click Removing To quickly remove all inactive traces from the window: inactive traces 2. Select Remove Inactive Traces from the Display menu. 2.4.6 Expanding and Linking Traces When you have more than one trace displayed in a window, you can: Click the trace to remove.

  • Page 78: Recalling And Rearranging Traces (Processing History)

    Chapter 2 Using Chromatogram and Spectrum Windows 2.4.7 Recalling and Rearranging Traces (Processing History) Overview Recalling or rearranging Removing traces from the history list 2-22 Applied Biosystems 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.
  • Page 79 Setting To set Processing History options: Processing From the Tools menu, select Processing History History options Options. The Processing History Options dialog box (Figure 2-10) is displayed. Figure 2-10 Processing History Options Dialog Box 2. Turn Processing History on or off. If you turn on, specify: 3.

  • Page 80: Overlaying Traces

    Chapter 2 Using Chromatogram and Spectrum Windows 2.4.8 Overlaying Traces Overlaying traces from different data files 2-24 Applied Biosystems This section includes: • Overlaying traces from different data files • Overlaying traces in a single data file • Changing the active trace •...
  • Page 81 Overlaying traces To overlay traces in a single data file: in a single data file 2. To use settings other than defaults, set attributes for the 3. Click the trace of interest to activate it. 4. From the Display menu, select Overlay Traces. NOTE: When saving results, only the results for the active trace are saved.
  • Page 82 Chapter 2 Using Chromatogram and Spectrum Windows Changing the active trace Sequentially activating overlaid spectra Setting overlay attributes 2-26 Applied Biosystems To change the active trace in an overlay: From the Display menu, deselect Overlay. 2. Click the trace to activate. 3.
  • Page 83 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 •...

  • Page 84: Annotating Traces

    • Section 5.3, Manual Calibration, or Section 5.4, Automatic Calibration • Section 5.6, Mass Deconvolution (Mariner Data Only) • Section 6.2, Using the Isotope Calculator • Section 6.3, Using the Mass Resolution Calculator • Section 6.4, Using the Signal-to-Noise Ratio Calculator 2.
  • Page 85 Annotating the To annotate the trace: trace Click the trace at the location where you want to insert text. 2. Right-click, then select: 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.

  • Page 86: Viewing Trace Labels

    Chapter 2 Using Chromatogram and Spectrum Windows 2.4.10 Viewing Trace Labels Chromatogram trace labels Chromatogram Trace Label Mass xxx:yyy + TAC 2-30 Applied Biosystems 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.
  • Page 87 Chromatogram Trace Label + 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”...
  • Page 88 Chapter 2 Using Chromatogram and Spectrum Windows Spectrum Trace Label DECONV (Mariner data only) RSMX (does not apply to Voyager PSD data) SC (Mariner data only) Stitched PSD 2-32 Applied Biosystems Type of Processing Base peak intensity Centroid Diode array data Zero charge deconvoluted trace Deisotoped trace Isotope...

  • Page 89: Printing Traces

    Figure 2-12 illustrates a smoothed spectrum with an “SM5” spectrum trace label. 2.4.11 Printing Traces Printing traces To print traces: 2. Click the window (Chromatogram or Spectrum) to print. Trace label Figure 2-12 Spectrum Trace Label 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...
  • Page 90 Chapter 2 Using Chromatogram and Spectrum Windows 2-34 Applied Biosystems 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.

  • Page 91: Print Setup

    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 To dedicate the printer to landscape orientation: printer to From the Windows desktop, click Start, then select landscape...

  • Page 92: Working With Multiple Data Files

    Chapter 2 Using Chromatogram and Spectrum Windows 2.5 Working with Multiple Data Files 2.5.1 Working with Separate Data Files 2-36 Applied Biosystems When you have multiple data files open, you can: • Work with the data files separately to view, zoom, and print •...

  • Page 93: Copying Traces From Multiple Data Files To A Window

    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. From the Edit menu, select Copy, then Trace Data, or 3.

  • Page 94: Saving, Opening, And Deleting .Dat Results

    Chapter 2 Using Chromatogram and Spectrum Windows Comparing copied traces 2.6 Saving, Opening, and Deleting .DAT Results Saving results for .DAT files Opening results for .DAT files Deleting results for .DAT files 2-38 Applied Biosystems 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...

  • Page 95: Exporting, Opening, And Deleting .Rcd And .Rsd Results Files (Mariner Data Only)

    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 To export results to a .RSD or .RCD file: for .RCD and 1. With a data file open in Data Explorer, select a .RSD files 2.

  • Page 96: Saving, Opening, And Deleting .Spc Results Files (Mariner Data Only)

    Chapter 2 Using Chromatogram and Spectrum Windows Deleting results for .RCD and .RSD files 2.8 Saving, Opening, and Deleting .SPC Results Files (Mariner Data Only) Saving results for .SPC files Opening results for .SPC files 2-40 Applied Biosystems Use Windows NT Explorer to delete .RCD and .RSD result files.
  • Page 97 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.
  • Page 98 Chapter 2 Using Chromatogram and Spectrum Windows 2-42 Applied Biosystems...

  • Page 99: Peak Detection And Labeling

    3 Peak Detection and Labeling This chapter contains the following sections: Overview ... 3-2 Peak Detection ... 3-6 Peak List ... 3-37 Deisotoping a Spectrum... 3-45 Peak Labeling ... 3-52 Process that Occurs During Peak Detection, Centroiding, and Integration ... 3-67 Default Peak Detection Settings ...

  • Page 100: Chapter 3 Peak Detection And Labeling

    Chapter 3 Peak Detection and Labeling 3.1 Overview 3.1.1 Default Peak Detection Overview Applied Biosystems This section includes: • Default peak detection • The resolution-based peak detection routine When you open a data file, it is automatically peak detected. For: •...

  • Page 101: The Resolution-Based Peak Detection Routine

    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 The resolution-based peak detection routine applies to spectral data only. You can enable or disable the affected resolution-based peak detection routine as described on page 3-14.

  • Page 102: Detection Ranges

    Chapter 3 Peak Detection and Labeling Detection ranges Number of data points 41 47 53 Figure 3-1 Example Detection Ranges Calculated by Software Based on Number Applied Biosystems The software uses the following formula to calculate the expected number of data points in a peak: Expected data point corresponds number of...
  • Page 103 Overview Overlapping peak To accommodate spectral peaks that occur on the boundary of two peak detection ranges, the software creates detection detection ranges ranges that overlap (Figure 3-2). Peak A Peak B Peak C Peak D Range 4 Range 3 Range 2 Range 1 Figure 3-2 Overlapping Detection Ranges...

  • Page 104: Peak Detection

    Chapter 3 Peak Detection and Labeling 3.2 Peak Detection 3.2.1 Strategy for Mariner Peak Detection Strategy Applied Biosystems This section includes: • Strategy for Mariner peak detection • Strategy for Voyager peak detection • Setting peak detection parameters • Peak detection parameter descriptions •...

  • Page 105: Section 3.2.3, Setting Peak Detection Parameters

    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.

  • Page 106: Strategy For Voyager Peak Detection

    50% from the default resolution setting, fine-tune the Resolution setting and reapply peak detection. For more information, see Section 6.3, Using the Mass Resolution Calculator. 2. To aid in peak interpretation, do all of the following: • Baseline-correct—The Centroid% peak detection value is derived from a percentage of the peak height, which is measured from 0.
  • Page 107 3. If peak detection is not acceptable, keep the Use Problem High-mass peaks not Decrease Mass Resolution setting. The default Mass detected Resolution settings are optimized for masses below 20,000 Da. Noise detected as peaks Increase the %Max Peak Area. Decrease the Resolution.
  • Page 108 Chapter 3 Peak Detection and Labeling Problem Partially resolved peaks not detected 3-10 Applied Biosystems Suggested Actions 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.

  • Page 109: Setting Peak Detection Parameters

    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 • Setting Advanced Settings (spectrum data only) • Resetting Basic Settings Before you begin Before setting peak detection, read: •...
  • Page 110 Chapter 3 Peak Detection and Labeling 3-12 Applied Biosystems Figure 3-3 Chromatogram Peak Detection Setup Dialog Box 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.
  • Page 111 Setting Basic Basic Settings should provide acceptable peak detection for most applications. After you apply these parameters, no Settings further adjustment should be required. If further adjustment is (spectrum data) required, select Use Advanced Settings and adjust parameters as needed. To set Basic Settings parameters for spectrum data: Click the Spectrum window to activate it.
  • Page 112 Chapter 3 Peak Detection and Labeling 3-14 Applied Biosystems Figure 3-4 Spectrum Peak Detection Setup— Basic Settings Tab 5. If you are detecting PSD data, or want to override the Global Thresholds, select Use Advanced Settings and skip to step 7. 6.
  • Page 113 Defaults are listed in “Basic Settings (spectrum data only)” on page 3-22. NOTE: The Mass Resolution you set here is also used by the Elemental Composition Calculator, the Elemental Targeting Application, and the Default Smoothing function. For more information, see Section 6.1, Using the Elemental Composition Calculator, Section 6.6,...
  • Page 114 Chapter 3 Peak Detection and Labeling Setting Peak Processing parameters (spectrum data only) 3-16 Applied Biosystems To set Peak Processing parameters: Click the Peak Processing tab in the Spectrum Peak Detection Setup dialog box. The Peak Processing tab is displayed (Figure 3-5). Figure 3-5 Spectrum Peak Detection Setup—...

  • Page 115: Setting Advanced

    Setting Advanced To set Advanced Settings that you can apply locally to a selected detection range, and that override the thresholds set Settings in the Basic Settings tab: (spectrum data only) 2. Click the Advanced Settings tab in the Spectrum Peak In the Basic Settings tab, select Use Advanced Settings.
  • Page 116 Chapter 3 Peak Detection and Labeling Resetting Basic Settings 3-18 Applied Biosystems 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.

  • Page 117: Peak Detection Parameter Descriptions

    3.2.4 Peak Detection Parameter Descriptions This section describes: • Chromatogram settings • Basic Settings (spectrum data) • Peak Processing parameters (spectrum data only) • Advanced Settings (spectrum data only) Chromatogram Table 3-1 describes the parameters in the Chromatogram Peak Detection Setup dialog box (see Figure 3-3 on settings page 3-12).
  • Page 118 Chapter 3 Peak Detection and Labeling Table 3-1 Chromatogram Settings (Continued) Parameter Detection Ranges (continued) %Base Peak Intensity NOTE: This parameter was previously named Peak Threshold% and Base Peak Relative%. %Max Peak Area Filter Width 3-20 Applied Biosystems Description To delete a range, select the range, then click 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.
  • Page 119 Table 3-1 Chromatogram Settings (Continued) Parameter Filter Width Hint: Set Filter Width to a number equal to the number of points across the peak. To determine the number of points (continued) across a peak, you can change the trace display from Line to Vertical Bars.
  • Page 120 Chapter 3 Peak Detection and Labeling Basic Settings (spectrum data only) Table 3-2 Basic Settings Tab Parameters (Spectrum Data Only) Parameter Global Thresholds %Base Peak Intensity NOTE: This parameter was previously named Peak Threshold% and Base Peak Relative%. 3-22 Applied Biosystems 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).
  • Page 121 Table 3-2 Basic Settings Tab Parameters (Spectrum Data Only) (Continued) Parameter %Max Peak Area 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.
  • Page 122 NOTE: The Mass Resolution you set here is also used by the Elemental Composition Calculator, the Elemental Targeting Application, and the Default Smoothing function. For more information, see Section 6.1, Using the Elemental Composition Calculator, Section 6.6, Using the Elemental Targeting Application, and Section 5.7, Noise Filtering/Smoothing. Continued...
  • Page 123 Table 3-2 Basic Settings Tab Parameters (Spectrum Data Only) (Continued) Parameter Trace Settings Use same settings Applies settings to all traces in the active window. for all traces in view 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.
  • Page 124 Chapter 3 Peak Detection and Labeling Peak Processing parameters (spectrum data only) Table 3-3 Peak Processing Parameters (Spectrum Data Only) Parameter Integration Baseline Settings NOTE: You can set peak labels to display peak start, peak end, and baseline marks. See Section 3.5.2, Setting Chromatogram and Spectrum Peak Labels. Valley-to-Baseline Valley-to-Valley Spectrum Parameters...
  • Page 125 Table 3-3 Peak Processing Parameters (Spectrum Data Only) (Continued) Parameter Charge State Determination (Spectrum only) Maximum Charge Determines the peak spacing evaluated for the presence of State 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) ×...

  • Page 126: Chapter 3 Peak Detection And Labeling

    Chapter 3 Peak Detection and Labeling Advanced Settings (spectrum data only) Table 3-4 Advanced Settings (Spectrum Data Only) Parameter Peak Detection Settings Detection Ranges 3-28 Applied Biosystems 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).
  • Page 127 Table 3-4 Advanced Settings (Spectrum Data Only) (Continued) Parameter Detection Ranges You can set multiple, non-contiguous ranges and define (continued) 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: •...
  • Page 128 Chapter 3 Peak Detection and Labeling Table 3-4 Advanced Settings (Spectrum Data Only) (Continued) Parameter 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 %Max Peak Area Minimum Intensity...

  • Page 129: Section 1.4, Customizing The Data Explorer Window

    Table 3-4 Advanced Settings (Spectrum Data Only) (Continued) Parameter 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.

  • Page 130: Charge State Determination And Examples

    Chapter 3 Peak Detection and Labeling 3.2.5 Charge State Determination and Examples Isotopes Charge state determination Charge state parameter examples 3-32 Applied Biosystems 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: •...
  • Page 131 Max Charge State When the Max Charge State and Max Isotope # are set correctly (in the example shown in Figure 3-7, both are set and Max Isotope # to 4), the neurotensin 558 m/z isotope cluster contains four set correctly peaks at charge state 3.
  • Page 132 Chapter 3 Peak Detection and Labeling 3-34 Applied Biosystems 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).
  • Page 133 Peak Detection Max Isotope # If the Max Isotope # is set too low, the software incorrectly groups peaks into different isotope clusters. In this example set too low 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).
  • Page 134 Chapter 3 Peak Detection and Labeling Effect of Minimum Intensity Charge state determination troubleshooting 3-36 Applied Biosystems 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.

  • Page 135: Peak List

    3.3 Peak List This section describes: 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. Displaying To display the peak list, select Output Window from the View menu, then click the Chro Peak List or Spec Peak List tab at the bottom of the window (Figure 3-14).
  • Page 136 Chapter 3 Peak Detection and Labeling Contents of peak list Chromatogram peak list Spectrum peak list 3-38 Applied Biosystems The peak lists contain the following information. Each entry represents one chromatographic peak in the trace and includes: • Index, a sequential number assigned to each entry in the peak list •...

  • Page 137: Inserting Peaks In The Peak List

    3.3.2 Inserting Peaks in the Peak List Description 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. Procedure To insert peaks: 2.

  • Page 138: Saving The Peak List

    Chapter 3 Peak Detection and Labeling 3.3.3 Saving the Peak List Saving as a stand-alone .PKT file 3-40 Applied Biosystems 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.
  • Page 139 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.

  • Page 140: Sorting, Filtering, And Printing The Peak List

    Chapter 3 Peak Detection and Labeling 3.3.4 Sorting, Filtering, and Printing the Peak List Sorting the peak list Filtering the spectrum peak list 3-42 Applied Biosystems 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).
  • Page 141 2. Select Enable Peak List Filter, then select: Filter Type 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.
  • Page 142 Chapter 3 Peak Detection and Labeling Printing the peak list Deleting items from the peak list 3-44 Applied Biosystems 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.

  • Page 143: Deisotoping A Spectrum

    3.4 Deisotoping a Spectrum This section includes: • Description • During peak deisotoping • When to use • Requirements • Using the Deisotope function • Troubleshooting • Example • Returning to the original spectrum Description The Deisotope function reduces a spectrum to a centroided plot by deconvoluting the monoisotopic peaks from the current peak list.

  • Page 144: When To Use

    Chapter 3 Peak Detection and Labeling When to use 3-46 Applied Biosystems 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...

  • Page 145: Deisotoping A Spectrum

    Peaks in original trace If first three peaks are part of the same isotope cluster Contribution to expected isotope ratio by contaminant If first three peaks are part of the same isotope cluster, but contaminant is also present If two isotope clusters are present Figure 3-17 Interpreting a Deisotoped Trace Requirements...
  • Page 146 Chapter 3 Peak Detection and Labeling Using the Deisotope function 3-48 Applied Biosystems To use the Deisotope function: 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.
  • Page 147 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.
  • Page 148 Chapter 3 Peak Detection and Labeling Example Figure 3-19 Spectrum Before Deisotoping 3-50 Applied Biosystems 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. 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.
  • Page 149 Returning to the To return to the original spectrum: original spectrum • If the original spectrum was an unprocessed spectrum, • If the original trace was a processed spectrum, select select Spectrum Number from the Display menu. The number of the original spectrum is displayed in the Select Spectrum dialog box.

  • Page 150: Peak Labeling

    Chapter 3 Peak Detection and Labeling 3.5 Peak Labeling Factors affecting peak labeling Labeling peaks manually 3-52 Applied Biosystems This section includes: • Charge state labels • Setting chromatogram and spectrum peak labels • Setting custom peak labels Peak labels are displayed only for detected peaks in the peak list (displayed in the Output window).

  • Page 151: Charge State Labels

    3.5.1 Charge State Labels Charge state A unique feature of the Data Explorer software is the ability to label multiply charged isotope peaks with their charge state. labels 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.

  • Page 152: Setting Chromatogram And Spectrum Peak Labels

    Chapter 3 Peak Detection and Labeling 3.5.2 Setting Chromatogram and Spectrum Peak Labels Customizing colors, font, and size Setting chromatogram labels 3-54 Applied Biosystems • Isotopes must be resolved • Peak detection parameters must be set to detect all of the peaks in the isotope cluster •...
  • Page 153 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.
  • Page 154 Chapter 3 Peak Detection and Labeling Setting spectrum labels 3-56 Applied Biosystems 8. Click OK. The trace is displayed. The detected peaks that meet the peak labeling criteria are labeled. To label spectrum peaks: Click the Spectrum window to activate it. 2.
  • Page 155 5. Select the Mass Type (peak apex or peak centroid). 6. Select the Peak Mass Label Type: Label Mass 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.
  • Page 156 Chapter 3 Peak Detection and Labeling 3-58 Applied Biosystems 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.

  • Page 157: Deleting Labels

    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”...
  • Page 158 Chapter 3 Peak Detection and Labeling Charge state not displayed 3-60 Applied Biosystems 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. •...

  • Page 159: Setting Custom Peak Labels

    3.5.3 Setting Custom Peak Labels This section includes: • 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 Description A custom peak label displays the label name you enter.
  • Page 160 Chapter 3 Peak Detection and Labeling 3-62 Applied Biosystems Figure 3-23 User Label Setup Dialog Box 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.
  • Page 161 6. To manually enter label settings, click Figure 3-24 User Label Entry Dialog Box (Spectrum) 7. Set the following parameters: Spectrum Parameter Label Peak Mass (if Label Type selected is Mass) Mass Difference (if Label Type selected is Difference) Mass Tolerance Charge State The User Label Entry dialog box is displayed (Figure 3-24).
  • Page 162 Chapter 3 Peak Detection and Labeling Chromatogram Parameter Label X Value X Tolerance 3-64 Applied Biosystems Text of the label to display. Retention time or Spectrum Number of the peak to label. The default units for X Tolerance correspond to the units of the x-axis.
  • Page 163 Peak Labeling Applying user You can apply labels you previously created and saved in .LBC or .LBS files. labels from .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.
  • Page 164 Chapter 3 Peak Detection and Labeling User labels not displayed 3-66 Applied Biosystems 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. •...

  • Page 165: Process That Occurs During Peak Detection, Centroiding, And Integration

    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.
  • Page 166 Chapter 3 Peak Detection and Labeling 3-68 Applied Biosystems • 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.
  • Page 167 Process that Occurs During Peak Detection, Centroiding, and Integration After peak After peaks are detected: detection • Centroid mass is calculated for spectral data, then modified by Gaussian peak fitting, if it is selected. • Chromatographic and spectral peaks are integrated. •...
  • Page 168 Chapter 3 Peak Detection and Labeling Integration Valley-to-Baseline Figure 3-25 Valley-to-Baseline and Valley-to-Valley Integration 3-70 Applied Biosystems 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): •...

  • Page 169: 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 Default peak detection settings are contained in the following .SET files: provided • MARINER.SET • VOYAGERLINEAR.SET • VOYAGERREFLECTOR.SET •...
  • Page 170 Chapter 3 Peak Detection and Labeling Parameter Basic Settings (spectrum data) %Base Peak Intensity %Max Peak Area Use Resolution Dependent Settings Mass Resolution Peak Processing Integration Baseline Setting Centroid% Max. Charge State Max. Isotopes# Min. Intensity% Max. Intensity% 3-72 Applied Biosystems The following table lists the default settings in the .SET files provided for spectra.
  • Page 171 Parameter Advanced Detection Range Filter Width Increment Noise Threshold %Base Peak Intensity %Max Peak Area Min. Intensity Min. Area Additional Additional .SET files that have been developed for detection of different types of data are included in the Voyager .SET files C:\VOYAGER\SETTINGS directory.
  • Page 172 Chapter 3 Peak Detection and Labeling 3-74 Applied Biosystems...
  • Page 173 4 Examining Chromatogram Data This chapter contains the following sections: Overview ... 4-2 Creating an Extracted Ion Chromatogram ... 4-5 Creating an Extracted XAC Chromatogram (Mariner Data Only) ... 4-13 Noise Filtering/Smoothing ... 4-17 Adding and Subtracting Spectra ... 4-20 Displaying MS Method Data (Mariner Data Only) ...

  • Page 174: Overview

    Chapter 4 Examining Chromatogram Data 4.1 Overview Types of Mariner data Select Total Ion Chromatogram, which includes the entire mass range saved in the data file. Each data point represents the sum of all ion intensities in the corresponding spectrum. Base Peak Intensity trace, which includes only the base peak in each spectrum.
  • Page 175 You can display extracted chromatograms from Mariner data files by selecting the Process menu with a Chromatogram window displayed, then selecting: Select Extracted Ion— Extracted Ion Chromatogram (XIC), which includes only the Center Window signal response from a mass window or range. For more or Range information, see Section 4.2.1, Creating an Extracted Ion Chromatogram (XIC).
  • Page 176 Chapter 4 Examining Chromatogram Data Select Center Window Extracted Ion chromatogram, which includes only the response or Range from a mass window or range. For more information, see (XIC) Section 4.2.1, Creating an Extracted Ion Chromatogram (XIC). Neutral Loss Constant Neutral Loss Chromatogram, which extracts only the (CNL) response from a mass difference from a selected peak.

  • Page 177: 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 •...
  • Page 178 Chapter 4 Examining Chromatogram Data Applied Biosystems 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.
  • Page 179 Creating an Extracted Ion Chromatogram Figure 4-1 Extracted Ion Chromatogram Dialog Box 5. 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 6.
  • Page 180 Chapter 4 Examining Chromatogram Data From the Spectrum window Applied Biosystems 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.

  • Page 181: Creating A Constant Neutral Loss (Cnl) Chromatogram

    4.2.2 Creating a Constant Neutral Loss (CNL) Chromatogram This section includes: • Overview • Applications • Labeling spectrum peaks with mass difference (optional) • Procedure • Example Overview 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.
  • Page 182 Chapter 4 Examining Chromatogram Data Labeling spectrum peaks with mass difference (optional) Procedure 4-10 Applied Biosystems 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.
  • Page 183 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.
  • Page 184 Chapter 4 Examining Chromatogram Data Example Figure 4-4 TIC for Flavonoid Mixture Containing Three Peaks 4-12 Applied Biosystems 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.

  • Page 185: Creating An Extracted Absorbance Chromatogram (Xac) (Mariner Data Only)

    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 To create an extracted absorbance chromatogram (XAC) for a selected wavelength window or range:...
  • Page 186 Chapter 4 Examining Chromatogram Data 4-14 Applied Biosystems Figure 4-6 Extracted Absorbance Chromatogram Dialog Box 6. Specify the Extraction Mode: • Accumulative—Creates a single trace combining intensities of all specified wavelengths • Individual—Creates one trace for each specified wavelength 7. Click OK. The extracted absorbance chromatograms are displayed in the Chromatogram window with an XAC trace label.
  • Page 187 Creating an Extracted Absorbance Chromatogram (XAC) (Mariner Data Only) From the To create an extracted absorbance chromatogram for a wavelength range from the Spectrum window: Spectrum window 1. Click the Chromatogram window to activate it. 2. From the Display menu, select Traces, then select a 3.
  • Page 188 Chapter 4 Examining Chromatogram Data Figure 4-7 Extracted Absorbance Chromatogram 7. To return to the original trace, see “Returning to the original trace” on page 4-4. 4-16 Applied Biosystems...

  • Page 189: 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.
  • Page 190 Chapter 4 Examining Chromatogram Data Type of Data Higher resolution data Higher molecular weight data 4-18 Applied Biosystems 2. 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...
  • Page 191 Suggested Type of Data Method High-resolution data Noise Removal (NR) (Does not affect peak resolution.) 3. Click OK. 4. To return to the original trace, see “Returning to the 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...

  • Page 192: Adding And Subtracting Raw Spectra Within A Data File

    Chapter 4 Examining Chromatogram Data 4.5 Adding and Subtracting Raw Spectra Within a Data File Adding and subtracting spectra 4-20 Applied Biosystems 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.
  • Page 193 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.
  • Page 194 Chapter 4 Examining Chromatogram Data 4-22 Applied Biosystems 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.

  • Page 195: Displaying Ms Method Data (Mariner Data Only)

    4.6 Displaying MS Method Data (Mariner Data Only) Overview 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. In this section This section includes: •...
  • Page 196 Chapter 4 Examining Chromatogram Data Filtering event tags 4-24 Applied Biosystems 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.
  • Page 197 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.
  • Page 198 Chapter 4 Examining Chromatogram Data Evaluating filtered traces Displaying additional filtered traces 4-26 Applied Biosystems 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.

  • Page 199: 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.
  • Page 200 Chapter 4 Examining Chromatogram Data 4-28 Applied Biosystems 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.

  • Page 201: Using Baseline Correction

    4.7.2 Using Baseline Correction Description 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. When to use Baseline-correct if you are analyzing data: • With a baseline that is not flat, and you are using the •...

  • Page 202: Using Uv Trace Offset (Mariner Data Only)

    Chapter 4 Examining Chromatogram Data 4.8 Using UV Trace Offset (Mariner Data Only) 4-30 Applied Biosystems To align a UV trace with a chromatogram trace: 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.
  • Page 203 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.
  • Page 204 Chapter 4 Examining Chromatogram Data 4-32 Applied Biosystems...
  • Page 205 5 Examining Spectrum Data This chapter contains the following sections: Overview ... 5-2 Creating a Combined Spectrum ... 5-4 Manual Calibration... 5-5 Automatic Calibration... 5-26 Centroiding ... 5-36 Mass Deconvolution (Mariner Data Only) ... 5-37 Noise Filtering/Smoothing ... 5-42 Adjusting the Baseline ... 5-45 Truncating a Spectrum...

  • Page 206: Overview

    Chapter 5 Examining Spectrum Data 5.1 Overview Types of spectra you can display Voyager PSD spectra Creating macros to combine processing functions Applied Biosystems You can display the following types of spectrum data: • Single spectrum—Double-click any point in the TIC to display the corresponding spectrum.
  • Page 207 Returning to the Many processing functions generate a new trace. If you have Trace Replace mode set to Replace, the new trace replaces original spectrum the original trace. For information on Replace mode, see Section 2.4.4, Adding Traces from the Same Data File to a Window.

  • Page 208: Chapter 5 Examining Spectrum Data

    Chapter 5 Examining Spectrum Data 5.2 Creating a Combined Spectrum Applied Biosystems 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.

  • Page 209: 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.
  • Page 210 Chapter 5 Examining Spectrum Data Calibration references files (.REF) provided When to use manual calibration Accurate mass measurements Applied Biosystems 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.

  • Page 211: Manually Calibrating

    5.3.2 Manually Calibrating This section describes: • Before calibrating Voyager data • Manually calibrating a single spectrum • Applying new constants to the data file • Exporting calibration constants (.CAL file) • Applying new constants to additional files Before calibrating Before you calibrate Voyager data, do the following to improve mass accuracy: Voyager data...
  • Page 212 Chapter 5 Examining Spectrum Data Manually calibrating a single spectrum Applied Biosystems 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: Click the Spectrum window to activate it, then select the spectrum trace of interest.
  • Page 213 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.

  • Page 214: Chapter 5 Examining Spectrum Data

    Chapter 5 Examining Spectrum Data Manually matching peaks 5-10 Applied Biosystems 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 •...
  • Page 215 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...
  • Page 216 Chapter 5 Examining Spectrum Data Eliminating data points 5-12 Applied Biosystems 9. Repeat step 7 and step 8 until all desired peaks are in the matched list. 10. To eliminate unacceptable data points from the calibration, do either of the following: •...
  • Page 217 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...
  • Page 218 Chapter 5 Examining Spectrum Data Calibration statistics Automatically matching peaks 5-14 Applied Biosystems 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.
  • Page 219 NOTE: If you are calibrating Mariner data, see “Ensuring that masses match during calibration” on page 5-24. Applying new To apply the calibration constants to the data: constants to the data file If you are Click calibrating Mariner data Apply (including Calibration MS Method...
  • Page 220 Chapter 5 Examining Spectrum Data Exporting calibration constants (.CAL file) Applying new constants to additional files 5-16 Applied Biosystems The calculated calibration constants can be exported to a .CAL file for use with other data files. You have two options for exporting: •...

  • Page 221: Creating Or Modifying A Calibration Reference File (.Ref)

    5. To save the calibration to the data file, select Mass If you are Select calibrating Mariner Apply Calibration data (including MS Method data) Voyager Apply Calibration data Apply to All NOTE: This button is displayed only if you are calibrating a Voyager multispectrum data file.
  • Page 222 Reference Mass dialog box (see Figure 5-3 on page 5-10). NOTE: You can also create a reference file using the Ion Fragmentation calculator. For more information, see “Creating a calibration reference file (.REF)” on page 6-30. To create and save a calibration reference file: From the Process menu, select Mass Calibration, then select Edit/Create Reference File.
  • Page 223 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.
  • Page 224 Chapter 5 Examining Spectrum Data Modifying a calibration reference file 5-20 Applied Biosystems 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).
  • Page 225 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.

  • Page 226: Reverting To Instrument Calibration

    Chapter 5 Examining Spectrum Data 5.3.4 Reverting to Instrument Calibration If you are calibrating Mariner Apply Calibration data (including MS Method data) 5-22 Applied Biosystems The Revert to Instrument Calibration function does the following: • Mariner data—Reapplies the original calibration constants used to acquire the data.
  • Page 227 If you are Select calibrating Voyager Apply Calibration data Apply to All NOTE: This button is displayed only if you are calibrating a Voyager multispectrum data file. The following occurs The current spectrum is calibrated and displayed with an MC trace label. The calibration constants are saved with the spectrum.

  • Page 228: Hints For Calibrating Mariner Data

    Chapter 5 Examining Spectrum Data 5.3.5 Hints for Calibrating Mariner Data Ensuring that masses match during calibration 5-24 Applied Biosystems 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.

  • Page 229: Hints For Calibrating Voyager Data

    5.3.6 Hints for Calibrating Voyager Data Importing a If you import a calibration, you must import a calibration generated from a data file that was acquired on the same calibration instrument using identical settings for the following instrument setting parameters: •...

  • Page 230: Automatic Calibration

    Chapter 5 Examining Spectrum Data 5.4 Automatic Calibration 5.4.1 Overview of Automatic Calibration During automatic calibration 5-26 Applied Biosystems 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.
  • Page 231 • Matches all peaks that meet the specified • If any points exceed the specified Max Outlier Automatic The Automatic Calibration function in Data Explorer is useful for quickly calibrating all spectra in a Mariner data file, after calibration for you prepare automatic calibration settings.

  • Page 232: When To Use

    Chapter 5 Examining Spectrum Data When to use Automatic calibration for Voyager data When to use 5-28 Applied Biosystems Use automatic calibration for Mariner data when you: • Have many spectra to calibrate • Know in advance what reference masses to use •...

  • Page 233: Importing And Specifying Automatic Calibration Settings

    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: 2.
  • Page 234 Chapter 5 Examining Spectrum Data Figure 5-7 Automatic Calibration Settings Dialog Box 5-30 Applied Biosystems 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.
  • Page 235 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.
  • Page 236 Chapter 5 Examining Spectrum Data 5-32 Applied Biosystems 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.
  • Page 237 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: Enter Fit Rejection parameters: Click Save Settings to save the automatic calibration settings (reference masses, matching criteria, and fit rejection parameters) as part of processing settings in the .DAT file.

  • Page 238: Chapter 5 Examining Spectrum Data

    Chapter 5 Examining Spectrum Data 5.4.3 Automatically Calibrating (Mariner Data Only) Automatically calibrating 5-34 Applied Biosystems This section includes: • Automatically calibrating • Applying new constants to the data file • Calibration results • Applying auto calibration settings to other files To automatically calibrate: Open the data file to calibrate.
  • Page 239 Applying new To save the calibration constants for each spectrum in the data file, select Apply Calibration from the Process menu. constants to the data file Calibration Automatic calibration results are displayed in the Output window (Figure 5-9). results For each spectrum in the data file, results include: •...

  • Page 240: Centroiding

    Chapter 5 Examining Spectrum Data 5.5 Centroiding 5-36 Applied Biosystems 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.

  • Page 241: 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.
  • Page 242 Chapter 5 Examining Spectrum Data 5-38 Applied Biosystems 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.
  • Page 243 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.
  • Page 244 Chapter 5 Examining Spectrum Data Converting to zero-charge 5-40 Applied Biosystems 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.
  • Page 245 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...

  • Page 246: Noise Filtering/Smoothing

    Chapter 5 Examining Spectrum Data 5.7 Noise Filtering/Smoothing Description Procedure 5-42 Applied Biosystems The Noise Filter/Smooth function includes four options for reducing noise in a spectrum trace: • Default smoothing • Noise-filtering • Smoothing by the Gaussian method • Noise removal To noise-filter or smooth the display: Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing.
  • Page 247 3. Select the method to use based on the type of data you Suggested Type of Data Method Noisy, low-resolution Default data smoothing (RSM) (May affect peak resolution.) High-resolution data, Noise but the Noise Filter (NF) Removal method may yield better (May affect results peak...
  • Page 248 Chapter 5 Examining Spectrum Data Type of Data High-resolution data 5-44 Applied Biosystems Suggested Method Noise Specify the number of standard deviations of Removal noise to remove. The software automatically (NR) calculates the average white noise for all frequencies across the spectrum, then removes the specified number of standard (Does not deviations of noise.

  • Page 249: 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.
  • Page 250 Chapter 5 Examining Spectrum Data 5-46 Applied Biosystems 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.

  • Page 251: Using Baseline Correction

    %Max Peak Area compensates for a rising or falling baseline. example, Centroid% peak detection value, or the Peak Height % in the Resolution calculator), are derived from a peak height measured from 0. Display the spectrum of interest. keep the original data displayed after processing.

  • Page 252: Using Advanced Baseline Correction

    Chapter 5 Examining Spectrum Data 5.8.3 Using Advanced Baseline Correction Description 5-48 Applied Biosystems This section includes: • Description • When to use • Correcting the baseline • General guidelines for setting parameters • Troubleshooting • Returning to the original spectrum The Advanced Baseline Correction feature corrects the baseline by: •...
  • Page 253 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.
  • Page 254 Chapter 5 Examining Spectrum Data Parameter Peak Width (at half height) 5-50 Applied Biosystems 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.
  • Page 255 Parameter Peak Width Set Peak Width according to the data you are correcting: (at half height) • (continued) • • • • To determine the number of points across a peak, change the trace display from Line to Vertical Bars (select Graphic Options from the Display menu, click Graph # Setup and change Line Type).
  • Page 256 Chapter 5 Examining Spectrum Data Parameter Flexibility 5-52 Applied Biosystems 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.
  • Page 257 Parameter Degree 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).
  • Page 258 Chapter 5 Examining Spectrum Data General guidelines for setting parameters Condition Baseline follows a rising peak cluster Baseline is gently curving Baseline rise should be ignored and treated as signal Peak cluster 5-54 Applied Biosystems Refer to the following table to determine how to set Advance Baseline Correction parameters and obtain the desired baseline correction.
  • Page 259 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.

  • Page 260: Truncating A Spectrum

    Chapter 5 Examining Spectrum Data 5.9 Truncating a Spectrum Description Truncating 5-56 Applied Biosystems 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 •...
  • Page 261 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.
  • Page 262 Chapter 5 Examining Spectrum Data Figure 5-19 Truncated Spectrum—Low Mass Gate Spike Eliminated 5-58 Applied Biosystems...

  • Page 263: 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.
  • Page 264 Chapter 5 Examining Spectrum Data 5-60 Applied Biosystems 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.
  • Page 265 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.

  • Page 266: Autosaturation Correction (Mariner Data Only)

    Chapter 5 Examining Spectrum Data 5.11 AutoSaturation Correction (Mariner Data Only) Function Hardware requirements 5-62 Applied Biosystems 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.
  • Page 267 AutoSaturation Correction (Mariner Data Only) Effect on Mariner The AutoSaturation Correction feature is not applied to .RST files saved from the Mariner Instrument Control Panel, even .RST files 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.

  • Page 268: Adding And Subtracting Raw Or Processed Spectra From The Same Or Different Data Files (Dual Spectral Trace Arithmetic)

    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) 5-64 Applied Biosystems 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.
  • Page 269 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 7. Select Add or Subtract for Operation. 8.
  • Page 270 Chapter 5 Examining Spectrum Data 5-66 Applied Biosystems...
  • Page 271 6 Using Tools and Applications This chapter contains the following sections: Using the Elemental Composition Calculator ... 6-2 Using the Isotope Calculator ... 6-13 Using the Mass Resolution Calculator ... 6-20 Using the Signal-to-Noise Ratio Calculator ... 6-23 Using the Ion Fragmentation Calculator ...

  • Page 272: Chapter 6 Using Tools And Applications

    You can also use the Elemental Composition calculator to determine the identity of fragment masses. To do so, set Peak Labels to display Mass Difference From Selected Peak, then specify the mass of the intact molecule.

  • Page 273: Elemental Composition Calculator

    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. The Elemental Composition Calculator dialog box (Figure 6-1) is displayed. Figure 6-1 Elemental Composition Calculator Using the Elemental Composition Calculator Dialog Box ™...
  • Page 274 Chapter 6 Using Tools and Applications Applied Biosystems 4. 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. •...
  • Page 275 The results of the calculation are displayed in the Elemental Analysis tab of the Output window (Figure 6-2). Figure 6-2 Elemental Composition Calculation Results in Output Window Using the Elemental Composition Calculator • “Adding new elements and setting limits” on page 6-9 •...
  • Page 276 Chapter 6 Using Tools and Applications If no results are displayed Applied Biosystems 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.

  • Page 277: Setting Limits

    Setting limits for existing elements • Adding new elements and setting limits • Setting limits for other result types in the Elemental Composition Calculator dialog box. The Limits dialog box is displayed (Figure 6-3). Figure 6-3 Element Limits Dialog Box ™ Data Explorer...
  • Page 278 NOTE: The software ignores changes you make to the individual isotope minimum and maximum values. Click OK. Change limits for other elements as needed, then click OK to return to the Elemental Composition Calculator.
  • Page 279 Element Limits in the Elemental Composition Calculator dialog box. The Limits dialog box is displayed (Figure 6-5). 2. Click Using the Elemental Composition Calculator Figure 6-5 Element Limits Dialog Box . The Periodic Table (Figure 6-6) is displayed. ™...
  • Page 280 Chapter 6 Using Tools and Applications Figure 6-6 Periodic Table 3. Click an element to select it and to display the Isotope dialog box (Figure 6-7). 6-10 Applied Biosystems...
  • Page 281 Using the Elemental Composition Calculator 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.
  • Page 282 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.

  • Page 283: Using The Isotope Calculator

    6.2 Using the Isotope Calculator Description Use the Isotope calculator to generate a theoretical isotope distribution. You can compare or overlay the theoretical distribution with your observed distribution. Using the Isotope To use the Isotope calculator: Calculator 1. Display the spectrum containing the observed isotope 2.
  • Page 284 Chapter 6 Using Tools and Applications 6-14 Applied Biosystems 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 Elemental Any element from the Periodic...
  • Page 285 • Resolving Power—Resolves peaks using the resolving power (M/ M) you enter. • PPM—Resolves peaks within the number of PPM (parts per million) you enter. Data Explorer Using the Isotope Calculator ™ Software User’s Guide 6-15...
  • Page 286 Chapter 6 Using Tools and Applications Add and subtract examples 6-16 Applied Biosystems 10. 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. Set the Threshold %. Signal intensity below this percent intensity is not included in the calculation or display.
  • Page 287 -cyclodextrin with +2 charge state (m/z 522) With these parameters specified Formula*—C42H60O30 Group type—deselected Charge—2 *Formula entry is case sensitive Data Explorer Using the Isotope Calculator The software performs this calculation – H) The software performs this calculation ™ Software User’s Guide...
  • Page 288 Chapter 6 Using Tools and Applications Evaluating traces Returning to the original spectrum 6-18 Applied Biosystems 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.
  • Page 289 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 If results are not calculated, you may have tried to remove a group that is not present in the formula.

  • Page 290: Using The Mass Resolution Calculator

    NOTE: If the Spectrum window is not active, Resolution Calculator does not appear on the Tools menu. 4. In the Resolution Calculator dialog box (Figure 6-12), set the percentage of peak height at which to calculate resolution. The default is 50%, which calculates the resolution at the full width/half maximum (FWHM) of a peak.
  • Page 291 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.
  • Page 292 Chapter 6 Using Tools and Applications Resolution calculator results 6-22 Applied Biosystems The result is displayed in the Output window (Figure 6-13). Figure 6-13 Resolution Calculator Results...

  • Page 293: Using The Signal-To-Noise Ratio Calculator

    To calculate a signal-to-noise ratio: signal-to-noise 1. Click the Spectrum window to activate it. ratio 2. From the Tools menu, select S/N Calculator. Using the Signal-to-Noise Ratio Calculator software automatically calculates the average noise across the spectrum. for calculation. For accurate results, this method requires a flat (non-rising) baseline that does not include peaks.
  • Page 294 Chapter 6 Using Tools and Applications 6-24 Applied Biosystems 3. 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.

  • Page 295: Using The Ion Fragmentation Calculator

    If the calculated fragments are present in the current data file, you can label fragments. The calculator includes a list of defined amino acids and residues that you can add to as needed. Using the Ion...
  • Page 296 Chapter 6 Using Tools and Applications Figure 6-15 Ion Fragmentation Calculator Dialog Box 6-26 Applied Biosystems 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.

  • Page 297: Setting Options

    To add the labels generated by this operation to the list of available user labels, select Append new labels to existing user label list. For more information, see Section 3.5.3, Setting Custom Peak Labels. 12. Click OK. Using the Ion Fragmentation Calculator ™ Data Explorer Software User’s Guide 6-27...

  • Page 298: Chapter 6 Using Tools And Applications

    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. 16. Click Induce Fragmentation. Results are displayed in the Ion Fragmentation Calculator dialog box (Figure 6-18).
  • Page 299 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. Using the Ion Fragmentation Calculator ™ Data Explorer Software User’s Guide...

  • Page 300: Reference File

    Chapter 6 Using Tools and Applications Labeling peaks Figure 6-19 Labeled Ion Fragmentation Peaks for Creating a calibration reference file (.REF) 6-30 Applied Biosystems Click Label Peaks. The ion peaks specified in the Options dialog box are labeled on the trace if they are present (Figure 6-19).

  • Page 301: Using The Elemental Targeting Application

    6.6 Using the Elemental Targeting Application Description 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.
  • Page 302 Chapter 6 Using Tools and Applications 6-32 Applied Biosystems Note the following when entering formulas: • 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.

  • Page 303: Displaying Results

    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. •...

  • Page 304: Using The Macro Recorder

    Chapter 6 Using Tools and Applications 6.7 Using the Macro Recorder Description Macros provided In this section 6.7.1 Before Using the Macro Recorder Importing macros provided Maximum number of macros 6-34 Applied Biosystems The Macro Recorder feature in Data Explorer allows you to set up multi-step tasks to execute automatically when you click a macro button.
  • Page 305 Location of All macros you record are stored in a file called DATAEXPLORER.VB6 in the C:\MARINER\PROGRAM or macros C:\VOYAGER directory. Displaying the If the macro toolbar (Figure 6-22) is not displayed: macro toolbar 2. Select Macros, then click Close. If a numbered macro button is disabled (gray), no macro has been assigned to it.
  • Page 306 Insert Peak (chromatogram) Processing History Options Customize Toolbar Customize ToolMenu Macro commands, including Automatic Macros Elemental Targeting Ion Fragmentation Calculator Functions you perform in the Output window, for example, sorting or copying the peak list, are not supported by the Macro Recorder.

  • Page 307: Recording A Macro

    6.7.2 Recording a Macro To record a macro: 2. From the Tools menu, select Record New Macro. 3. Type a name and a description if desired. 4. Click OK. 5. Select the commands you want to automate with the 6. From the Tools menu, select Stop Macro Recording. Open a data file.

  • Page 308: Assigning Macros To Buttons

    Chapter 6 Using Tools and Applications 6.7.3 Assigning Macros to Buttons Assigning a macro to a button 6-38 Applied Biosystems 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.

  • Page 309: Running A Macro

    De-assigning a To de-assign a macro from a button, select the macro button in the Assign Macro dialog box, then click De-assign. macro from a button 6.7.4 Running a Macro You can run a macro using a: Using a toolbar To run a macro using the toolbar button: button 2.
  • Page 310 Chapter 6 Using Tools and Applications If the macro contains a syntax error 6-40 Applied Biosystems 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, it may cause the Data Explorer software to close unexpectedly.

  • Page 311: Deleting A Macro

    6.7.5 Deleting a Macro To delete a macro: 2. Select the macro to delete from the list. 3. Click Delete. From the Tools menu, select Macros. The Macros dialog box (Figure 6-26) is displayed. Figure 6-26 Macros Dialog Box NOTE: Other buttons on this dialog box are for advanced editing.

  • Page 312: Advanced Macro Editing

    Chapter 6 Using Tools and Applications 6.7.6 Advanced Macro Editing Accessing the Visual Basic Editor Displaying scriptable objects Numbering sequence 6-42 Applied Biosystems 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.

  • Page 313: Importing Or Exporting Macros In Dataexplorer.vb6

    6.7.7 Importing or Exporting Macros in DATAEXPLORER.VB6 You can import macros into, or export macros from, the DATAEXPLORER.VB6 project for use in the Data Explorer software. Importing when When you install a new version of the Data Explorer software: new versions of Data Explorer software installed To make the new macros available for use, import them into...
  • Page 314 Chapter 6 Using Tools and Applications Exporting 6-44 Applied Biosystems 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.

  • Page 315: Running Macros Automatically When Opening And Closing Files

    6.7.8 Running Macros Automatically When Opening and Closing Files 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: 2.
  • Page 316 Chapter 6 Using Tools and Applications 6-46 Applied Biosystems...
  • Page 317 7 Data Explorer Examples This chapter contains the following sections: Mariner Data Examples ... 7-2 7.1.1 7.1.2 7.1.3 Voyager Data Examples... 7-11 7.2.1 7.2.2 7.2.3 Improving Signal-To-Noise Ratio ... 7-2 Deconvoluting and Evaluating Unresolved Chromatographic Peaks ... 7-4 Determining if a Peak is Background Noise ...

  • Page 318: Mariner Data Examples

    Chapter 7 Data Explorer Examples 7.1 Mariner Data Examples 7.1.1 Improving Signal-To-Noise Ratio Overview Creating an extracted ion chromatogram Applied Biosystems This section includes: • Improving signal-to-noise ratio • Deconvoluting and evaluating unresolved chromatographic peaks • Determining if a peak is background noise 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.
  • Page 319 Figure 7-1 illustrates the improved signal-to-noise ratio in the extracted ion chromatogram for three replicate loop injections. Original TIC containing all masses Figure 7-1 Improving Signal-To-Noise Ratio with an Extracted Ion Chromatogram Mariner Data Examples Extracted ion chromatogram for 609 Da with improved signal-to-noise ratio ™...

  • Page 320: Deconvoluting And Evaluating Unresolved Chromatographic Peaks

    Chapter 7 Data Explorer Examples 7.1.2 Deconvoluting and Evaluating Unresolved Chromatographic Peaks Overview Creating a combined spectrum Figure 7-2 Unresolved Peaks in Cytochrome C Applied Biosystems You can use the Data Explorer software to deconvolute chromatographic peaks and obtain masses for each component by: •...
  • Page 321 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.

  • Page 322: Unresolved Chromatographic Peaks

    Chapter 7 Data Explorer Examples Original TIC containing unresolved peaks Figure 7-4 Deconvoluting Unresolved Chromatographic Peaks Creating combined spectra Applied Biosystems Extracted ion chromatograms with deconvoluted peaks Create a combined spectrum for each extracted ion chromatogram: 1. Activate the Spectrum window, then click toolbar two times to add two traces.
  • Page 323 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.

  • Page 324: Determining If A Peak Is Background Noise

    Chapter 7 Data Explorer Examples 7.1.3 Determining if a Peak is Background Noise Overview Subtracting spectral peaks Applied Biosystems To determine if spectral peaks represent low-level components or if they are due to solvent contribution, you can: • Subtract the spectral peaks from the chromatogram •...
  • Page 325 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). Figure 7-6 Subtracting Spectra 6. Click OK. The subtracted spectrum is displayed (Figure 7-7 on page 7-10).
  • Page 326 Chapter 7 Data Explorer Examples Creating extracted ion chromatogram Figure 7-8 Extracted Ion Chromatogram for Mass 391 Da 7-10 Applied Biosystems Figure 7-7 Subtracted Spectrum The peak at 391 is still present, which indicates one of the following conditions: • You did not subtract sufficient baseline •...

  • Page 327: 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 7.2.1 Detecting and Labeling Partially Resolved Peaks If peaks are not If peaks are partially resolved and the peaks of interest are not labeled labeled, you can adjust the following peak detection parameters:...
  • Page 328 Chapter 7 Data Explorer Examples Figure 7-9 Partially Resolved Peaks That Represent Two Compounds, Minor Adjusting peak detection 7-12 Applied Biosystems Component Not Detected 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.
  • Page 329 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.

  • Page 330: Processing Before Calibrating To Optimize Mass Accuracy

    Chapter 7 Data Explorer Examples 7.2.2 Processing Before Calibrating to Optimize Mass Accuracy Calibrating without baseline correcting and deisotoping 7-14 Applied Biosystems This section includes: • Calibrating without baseline correcting and deisotoping • Before calibrating • Calibrating For optimum mass accuracy, baseline correct and deisotope a spectrum before calibrating.
  • Page 331 Before calibrating To optimize mass accuracy, do the following before calibrating: 1. Display the spectrum of interest. Baseline correcting 2. From the Process menu, select Baseline Correction. Deisotoping 3. From the Peaks menu, select Peak Deisotoping. 4. For this example spectrum, specify H for Adduct and 5.
  • Page 332 Chapter 7 Data Explorer Examples Calibrating 7-16 Applied Biosystems 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.
  • Page 333 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.

  • Page 334: Detecting Peaks From Complex Digests

    Chapter 7 Data Explorer Examples 7.2.3 Detecting Peaks from Complex Digests Overview 7-18 Applied Biosystems 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. •...
  • Page 335 Procedure To detect peaks from complex mixtures: 1. Display the spectrum of interest. Noise 2. From the Process menu, select Noise Filter/Smooth. filtering/smoothing 3. Select Default Smoothing or Noise Filter (with a 4. Click Setting detection thresholds The Noise Filter/Smooth dialog box (Figure 7-19) is displayed.
  • Page 336 Chapter 7 Data Explorer Examples Figure 7-20 Spectrum Peak Detection Setup— Basic Settings Tab 5. Click Use Advanced Settings. The Advanced Settings tab is displayed (Figure 7-21). 7-20 Applied Biosystems...
  • Page 337 Figure 7-21 Spectrum Peak Detection Setup— 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.
  • Page 338 Chapter 7 Data Explorer Examples Increasing detection thresholds 7-22 Applied Biosystems Figure 7-22 Deisotoping Dialog Box 10. For this example spectrum, specify H for Adduct and C6H5NO for Generic Formula. Click OK. For more information on deisotoping, see Section 3.4, Deisotoping a Spectrum. 12.
  • Page 339 8 Viewing Voyager PSD Data This chapter contains the following sections: Displaying PSD Data ... 8-2 Applying Fragment Labels ... 8-8 Calibrating a PSD Spectrum ... 8-10 8.3.1 8.3.2 8.3.3 8.3.4 8.3.5 Checking Peak Detection ... 8-11 Calibrating ... 8-12 Creating PSD .CAL Files and Applying to Other Data Files ...

  • Page 340: Chapter 8 Viewing Voyager Psd Data

    Chapter 8 Viewing Voyager PSD Data 8.1 Displaying PSD Data Displaying the composite spectrum Applied Biosystems 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.
  • Page 341 Advancing To advance through segment traces, click Segments are displayed in the order in which they were through segment acquired. traces NOTE: If these buttons are not displayed in the toolbar, you can add them. See Section 1.4.3, Customizing Toolbars. Displaying To display multiple segment traces: multiple segment...
  • Page 342 Chapter 8 Viewing Voyager PSD Data Applied Biosystems 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. Figure 8-2 PSD Processing Dialog Box The Max Stitch Mass is equal to the Precursor Mass times the Mirror Ratio.
  • Page 343 3. In the Spectrum window, click the first Not Used trace. 4. In the PSD Processing dialog box, double-click the Entry 5. Repeat step 3 and step 4 to display additional segments. 6. To advance through traces, select any trace, then click Redisplaying the To redisplay the composite spectrum, click Generate Composite in the PSD Processing dialog box (Figure 8-2).
  • Page 344 Chapter 8 Viewing Voyager PSD Data How the composite spectrum is generated PSD calibration equation Applied Biosystems 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.
  • Page 345 If you are performing an internal standard calibration, the software determines the constants as listed below: PSD Internal Standard Calibration One-point Two-point or three-point More than three-point Region of The composite spectrum is generated from portions of the segment traces. The upper mass limit of the composite region segments included in each segment is determined by the PSD Mirror Ratio (R in composite...

  • Page 346: Applying Fragment Labels

    8.2 Applying Fragment Labels Overview Applying labels Applied Biosystems 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. To apply fragment labels to PSD spectra: From the Applications menu, select Ion Fragmentation Calculator.

  • Page 347: Section 6.5, Using The Ion Fragmentation Calculator

    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.

  • Page 348: Calibrating A Psd Spectrum

    Chapter 8 Viewing Voyager PSD Data 8.3 Calibrating a PSD Spectrum When to use this procedure Overview of creating a PSD .CAL file 8-10 Applied Biosystems NOTE: Multi-point calibration yields higher mass accuracy than one-point calibration. This section includes: • Checking peak detection •...

  • Page 349: Checking Peak Detection

    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 •...

  • Page 350: Calibrating

    Chapter 8 Viewing Voyager PSD Data 8.3.2 Calibrating Calibrating 8-12 Applied Biosystems This section includes: • 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: Click the Spectrum window to activate it.

  • Page 351: Section 5.3.3, Creating Or Modifying A Calibration Reference File (.Ref)

    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 (.REF) file that specifies the peak type for reference masses as Resolved Isotope Mass (even if they are not resolved...
  • Page 352 Chapter 8 Viewing Voyager PSD Data 8-14 Applied Biosystems 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.
  • Page 353 Matching peaks If you want the software to compare observed masses in all segment spectra included in the .DAT file to reference masses automatically in the selected calibration reference file: 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.
  • Page 354 Chapter 8 Viewing Voyager PSD Data Selecting peaks manually 8-16 Applied Biosystems 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: Right-click-drag over the peak of interest.
  • Page 355 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.
  • Page 356 Chapter 8 Viewing Voyager PSD Data Solving and plotting Applying new constants to the data file 8-18 Applied Biosystems 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.
  • Page 357 Selecting To improve calibration statistics, you can select the same fragment ion from more than one segment. Monoamino acid calibration peaks fragments (immonium ions) below 150 Da are useful for this for optimum mass purpose. Because the segments have been collected with accuracy different PSD Mirror Ratios, the software allows you to add the same mass to the list multiple times.

  • Page 358: Creating Psd Calibration (.Cal) Files And Applying To Other Data Files

    Chapter 8 Viewing Voyager PSD Data 8.3.3 Creating PSD Calibration (.CAL) Files and Applying to Other Data Files Creating PSD .CAL files Applying new constants to additional files 8-20 Applied Biosystems To generate a PSD .CAL file: Acquire a standard, for example, angiotensin, in the Instrument Control Panel in PSD mode.

  • Page 359: Creating Psd Calibration Reference (.Ref) Files

    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.
  • Page 360 Chapter 8 Viewing Voyager PSD Data 8-22 Applied Biosystems 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).

  • Page 361: Changing The Precursor Mass

    8.3.5 Changing the Precursor Mass When to change When analyzing the composite spectrum, you may find that the observed fragments and sequence are not consistent with precursor mass 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.
  • Page 362 Chapter 8 Viewing Voyager PSD Data Changing 8-24 Applied Biosystems If the precursor mass taken from the data file is not correct: 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 •...

  • Page 363: Troubleshooting

    9 Troubleshooting This chapter contains the following sections: Overview ... 9-2 General Troubleshooting... 9-3 Processing, Tools, and Applications Troubleshooting ... 9-6 Calibration Troubleshooting ... 9-10 Printing Troubleshooting ... 9-14 Peak Detection and Labeling Troubleshooting ... 9-15 Chapter ™ Data Explorer Software User’s Guide...

  • Page 364: Chapter 9 Troubleshooting

    Chapter 9 Troubleshooting 9.1 Overview Applied Biosystems 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.

  • Page 365: General Troubleshooting

    9.2 General Troubleshooting Table 9-1 General Troubleshooting—Mariner and Voyager Symptom Cannot find data file Error message displayed when opening PSD data file Parts of other software windows are displayed on top of the Data Explorer window, or toolbar buttons or status indicators are not displayed “Failed to create empty document”...
  • Page 366 Chapter 9 Troubleshooting Table 9-1 General Troubleshooting—Mariner and Voyager (Continued) Symptom M/z range in data files converted to centroid does not match m/z range in original data file Text annotation from a previous trace displayed on current trace Table 9-2 General Troubleshooting—Mariner Only Symptom “Failed to open chromatogram data”...
  • Page 367 Table 9-2 General Troubleshooting—Mariner Only (Continued) Symptom Spectra labeled with You are viewing spectrum numbers that do event-filtered MS Method not correspond to the axis data. Spectra in an event-filtered trace are numbered contiguously (1,2,3...) regardless of their relation to the overall acquisition.

  • Page 368: Processing, Tools, And Applications Troubleshooting

    Troubleshooting 9.3 Processing, Tools, and Applications Troubleshooting Table 9-4 Processing, Tools, and Applications Troubleshooting—Mariner and Symptom Failed to calculate result for isotope calculator All traces in an overlaid trace are not processed Applied Biosystems Voyager Possible Cause You may have tried to...
  • Page 369 Table 9-4 Processing, Tools, and Applications Troubleshooting—Mariner and Symptom Results not saved for all Only results for the active traces in an overlaid trace trace are saved Link View command does You did not select Link not have an effect on View for each window or windows or open data files data file...
  • Page 370 Chapter 9 Troubleshooting Table 9-4 Processing, Tools, and Applications Troubleshooting—Mariner and Symptom After Single-charge Conversion of multiply charged peaks, you see charge states other than 0 or 1 Applied Biosystems Voyager (Continued) Possible Cause Peaks in the original 1. Set peak detection spectrum are labeled with an incorrect charge state 2.
  • Page 371 Table 9-5 Processing, Tools, and Applications Troubleshooting—Mariner Only Symptom Failed to calculate result for mass deconvolution Multiple Charge/Mass Deconvolution commands dimmed on Process menu Centroiding, Mass Calibration, Multiple Charge commands not displayed on Process menu Resolution command not displayed on Tools menu Only the active trace zooms in Overlay mode Processing, Tools, and Applications Troubleshooting...

  • Page 372: Calibration Troubleshooting

    Chapter 9 Troubleshooting 9.4 Calibration Troubleshooting Table 9-6 Calibration Troubleshooting—Mariner and Voyager Symptom Auto Calibration is turned on, but current spectrum is not auto calibrated During calibration, the software is not matching spectrum masses to some reference masses in the calibration reference file 9-10 Applied Biosystems...
  • Page 373 Table 9-6 Calibration Troubleshooting—Mariner and Voyager (Continued) Symptom Calibration returns an When creating a reference invalid number of matches 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...
  • Page 374 Chapter 9 Troubleshooting Table 9-7 Calibration Troubleshooting—Mariner Only Symptom Mass Calibration commands are dimmed Apply Calibration command is dimmed when calibrating MS Method data Error displayed when you import a calibration 9-12 Applied Biosystems Possible Cause Chromatogram window is Select Spectrum window. selected For MS Method data, No action.
  • Page 375 Table 9-8 Calibration Troubleshooting—Voyager Only Symptom Error displayed when you .CAL file corrupted import a calibration Importing a .CAL file generated from a Mariner data file Importing a .CAL file generated from a data file collected in a different instrument mode (Linear, Reflector, or PSD) Importing a .CAL file generated on a different...

  • Page 376: Printing Troubleshooting

    Print All Views before you select Print All from the File menu with Views. For more more than two data files information, see open. We have observed Section 2.4.11, Printing this on HP LaserJet4, Traces. HP LaserJet5, and HP LaserJet6 printers. Action...

  • Page 377: 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 Symptom Peaks are not detected or labeled Peak Detection and Labeling Troubleshooting Voyager Possible Cause Peaks are very close...

  • Page 378: Section 3.2.2, Strategy For Voyager Peak Detection

    Chapter 9 Troubleshooting Table 9-10 Peak Detection and Labeling Troubleshooting—Mariner and Voyager Symptom Peaks are not detected or labeled (continued) When creating a custom label for a spectrum, you right-click-drag across a peak to identify the peak, and an extracted ion chromatogram is created instead 9-16...
  • Page 379 Table 9-10 Peak Detection and Labeling Troubleshooting—Mariner and Voyager Symptom Expected user label not displayed Peak label placed on peak shoulder instead of peak apex Table 9-11 Peak Detection and Labeling Troubleshooting—Voyager Only Symptom Noise detected as peaks Low Mass Gate spike identified as Base Peak (linear data) Peak Detection and Labeling Troubleshooting...
  • Page 380 Chapter 9 Troubleshooting Table 9-11 Peak Detection and Labeling Troubleshooting—Voyager Only Symptom Partially resolved peaks not detected Table 9-12 Charge State and Isotope Determination Troubleshooting—Mariner Symptom Known isotope labeled with incorrect charge state (too low) 9-18 Applied Biosystems Possible Cause Mass resolution set too Decrease Mass high to detect average...
  • Page 381 Table 9-12 Charge State and Isotope Determination Troubleshooting—Mariner Symptom Known isotope not labeled with charge state Peak Detection and Labeling Troubleshooting Only (Continued) Possible Cause Charge State peak labels Turn on Charge State disabled peak labels. See Section 3.5.2, Setting Chromatogram and Spectrum Peak Labels.
  • Page 382 Chapter 9 Troubleshooting Table 9-12 Charge State and Isotope Determination Troubleshooting—Mariner Symptom Spectrum peaks not labeled with charge state when charge state labels are selected 9-20 Applied Biosystems Only (Continued) Possible Cause Mass of original molecule No action. Normal above 4,000 Da, not occurrence.
  • Page 383 Table 9-12 Charge State and Isotope Determination Troubleshooting—Mariner Symptom Spectrum peaks labeled with incorrect charge state when charge state labels are selected (continued) Monoisotopic peak not labeled correctly Peak Detection and Labeling Troubleshooting Only (Continued) Possible Cause Charge state Adjust parameters. See: determination parameters are set such that peaks are determined to have no...
  • Page 384 Chapter 9 Troubleshooting 9-22 Applied Biosystems...

  • Page 385: Limited Product Warranty

    A Warranty Limited Product Warranty Limited 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.

  • Page 386: Appendix A Warranty

    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.
  • Page 387 Warranties THE REMEDIES PROVIDED HEREIN ARE BUYER’S SOLE AND EXCLUSIVE REMEDIES. WITHOUT LIMITING THE limitations GENERALITY OF THE FOREGOING, IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTACT, TORT, WARRANTY OR UNDER ANY STATUTE (INCLUDING WITHOUT LIMITATION ANY TRADE PRACTICE, UNFAIR COMPETITION OR OTHER STATUTE OF SIMILAR IMPORT) OR ON ANY OTHER BASIS, FOR DIRECT, INDIRECT, PUNITIVE, INCIDENTAL,...
  • Page 388 Appendix A Warranty Applied Biosystems...
  • Page 389 Appendix 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 ... B-8 ™ Data Explorer Software User’s Guide...

  • Page 390: Overview

    Appendix B Overview of Isotopes B.1 Isotopes Overview Applied Biosystems 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 C, but natural carbon also contains Because a mass spectrometer measures mass-to-charge...
  • Page 391 As the number of carbon atoms in a compound increases, the possibility of the compound containing a also increases. A compound with ten carbon atoms includes a molecular ion M greater than the molecular ion, which is approximately 11 percent of the abundance of the molecular ion. The possibility of including two also increases with increasing number of carbon atoms.
  • Page 392 Appendix B Overview of Isotopes (M+1) (M+2) 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 C isotope peak exceeds the height of the C peak. Isotope-limited The mass range you analyze and the resolving power of the resolution...
  • Page 393 Isotopes (M+1) (M+2) 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).
  • Page 394 Appendix B Overview of Isotopes B.2 Monoisotopic and Average Masses Applied Biosystems 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 mass corresponds to lowest mass peak Figure B-6 Monoisotopic Mass When isotopes are not resolved (Figure B-7), the average...
  • Page 395 Monoisotopic and Average Masses Average mass corresponds to centroid of unresolved peak cluster Figure B-7 Average Mass ™ Data Explorer Software User’s Guide...
  • Page 396 Appendix B Overview of Isotopes B.3 Isotopes of Common Elements Table B-1 Isotopes of Common Elements Isotope Mass 1.0078 2.0141 13.0033 14.0030 15.0001 15.9949 16.9991 17.9991 18.9984 22.9897 27.9769 28.9764 29.9737 1. Meth. Enzymol., McCloskey, J.A, ed., 1990, 193. Applied Biosystems Table B-1 lists the natural abundance of isotopes for some common elements seen in mass spectrometry Natural...
  • Page 397 C Data Explorer Toolbox (Visual Basic Macros) Overview... C-2 Preparing Data Before Accessing Macros ... C-3 Accessing the Macros ... C-4 Using the Ladder Sequencing Toolbox ... C-5 Using the Peptide Fragmentation Toolbox ... C-9 Using the Polymer Analysis Toolbox ... C-15 Using MS Fit/MS Tag Toolbox ...

  • Page 398: C.1 Overview

    Appendix C Data Explorer Toolbox (Visual Basic Macros) C.1 Overview Macros provided Importing macros provided Modifying the macros Applied Biosystems The following toolbox of Visual Basic macros is provided with the Data Explorer software: • Ladder Sequencing Use when performing sequencing to label peaks with the appropriate amino acid, DNA residue, or RNA residue.

  • Page 399: Preparing Data Before Accessing Macros

    References The following references are selected by default in the required DataExplorerProject in the Visual Basic Editor and are required for the macros in the DataExplorer.VB6 file to successfully run: To view references: 2. Select References from the Tools menu in the Visual C.2 Preparing Data Before Accessing Macros Before you access the toolbox, open data files of interest and:...

  • Page 400: Accessing The Macros

    Appendix C Data Explorer Toolbox (Visual Basic Macros) C.3 Accessing the Macros Applied Biosystems To access the macros: 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.

  • Page 401: 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.
  • Page 402 Appendix C Data Explorer Toolbox (Visual Basic Macros) Applied Biosystems 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.
  • Page 403 Using the Ladder Sequencing Toolbox 7. 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 Difference Difference between the current peak and the reference, preceded by a minus sign (–).
  • Page 404 Appendix C Data Explorer Toolbox (Visual Basic Macros) Displaying the original labels Applied Biosystems If DNA or RNA is selected, the software: • 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.) •...

  • Page 405: Using The Peptide Fragmentation Toolbox

    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.
  • Page 406 Appendix C Data Explorer Toolbox (Visual Basic Macros) Pairs C-10 Applied Biosystems 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.
  • Page 407 • • 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.
  • Page 408 Appendix C Data Explorer Toolbox (Visual Basic Macros) C-12 Applied Biosystems 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.
  • Page 409 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, or print, as needed. Correlation To list ion pair correlations: Click the Correlation tab. 2.
  • Page 410 Appendix C Data Explorer Toolbox (Visual Basic Macros) Displaying the original labels C-14 Applied Biosystems 4. Click Find Correlation. Correlations for the selected peak are listed. NOTE: Yp, Sp and Tp represent phosphotyrosine, phosphoserine, and phosphothreonine, respectively. 5. Click Copy to Output Window Result Tab to copy results to the Result tab.

  • Page 411: 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: The Polydispersity Index represents how widely dispersed the polymeric distribution is. A lower value (for example, 1.02) indicates a narrowly dispersed polymer.
  • Page 412 Appendix C Data Explorer Toolbox (Visual Basic Macros) C-16 Applied Biosystems 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.
  • Page 413 • CAUTION: Correct labeling of the peaks is essential when using the labeled peaks option. 4. Click Calculate. 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.

  • Page 414: Using Ms Fit/Ms Tag Toolbox

    Appendix C Data Explorer Toolbox (Visual Basic Macros) C.7 Using MS Fit/MS Tag Toolbox Preparing data before accessing Running MS Fit/MS tag C-18 Applied Biosystems Use the MS Fit/MS Tag toolbox when analyzing protein digest, peptide, or peptide fragment spectra to perform a protein database search.
  • Page 415 2. Click: • MS-Fit tab If you are examining peptide data from a protein digest. • MS-Tag tab If 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.
  • Page 416 Appendix C Data Explorer Toolbox (Visual Basic Macros) C-20 Applied Biosystems...
  • Page 417 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...
  • Page 418 Amino acids, labeling C-5 Analog signal, displaying 4-2 Analyzer Temperature, displaying trace 4-2 ANGIOTENSIN_FRAGMENTS.REF 5- Annotating traces adding text 2-29 deleting text 2-29 text from previous trace displayed 2-29 with ASCII text 2-28 with results 2-28 Apex mass copying from peak list 1-41 labeling 3-57 Applied Biosystems Technical Support 9-2...
  • Page 419 DAT 1-36 importing 5-16, 8-20 importing, error displayed 9-11, 9-12 PSD, creating 8-20 PSD, overview of creating 8-10 saving 5-16 Calculator tools elemental composition 6-2 Ion Fragmentation 6-25 isotope 6-13 mass resolution 6-20 signal-to-noise ratio 6-23 Calibrating mass, automatic (Mariner...
  • Page 420 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...
  • Page 421 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...
  • Page 422 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,...
  • Page 423 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...
  • Page 424 Customizing (continued) SET files 1-17 toolbars 1-22 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...
  • Page 425 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...
  • Page 426 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...
  • Page 427 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.VB6 6-44 MSM and CAL from DAT 1-36 results 2-39 RSD and RCD files 2-39 trace to ASCII format 1-34...
  • Page 428 6-5 generating list of masses with Ion Fragmentation calculator 6-25 identifying with Elemental Composition calculator 6-2 labeling 6-25, C-9 Peptide fragmentation macro C-9 Fragment spectrum, PSD, see Segment spectrum, PSD FRM files for macros 6-43...
  • Page 429 3-21, 3-70 Valley-to-Valley, spectrum 3-26, 3-70 Internal mass calibration 5-5, 5-26 Internal standard calibration, see Calibrating mass Ion Fragmentation Calculator description 6-25 procedure 6-25 PSD segment spectra, labeling 8-8 REF file, PSD, creating 8-21 results 6-29 sequence codes, acceptable 6-26...
  • Page 430 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 Keywords, Windows NT entering 1-31 searching 1-32 viewing 1-32 Labeling peaks see also Peak labels chromatogram 3-54 customizing label appearance 1-25 factors affecting 3-52 manually 3-39...
  • Page 431 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.VB6 6-43 location of macros 6-35 maximum number of macros 6-34...
  • Page 432 3-57 Mass list, copying to Windows clipboard 1-41 Mass Offset, replaced by Mass Difference from Selected Peak 3-57 Mass Resolution Calculator, see Resolution, mass Mass resolution defaults used in peak detection 3-24 Match Charge State 3-62 Max Stitch Mass...
  • Page 433 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...
  • Page 434 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...
  • Page 435 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...
  • Page 436 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...
  • Page 437 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,...
  • Page 438 Related documents xiv Removing traces active 2-21 inactive 2-21 Replace mode, setting for added traces 2-18 Resolution Calculator not displayed 9-8 Resolution, mass calculating 6-20 command not on menu 9-8 default peak height used 6-20 defaults used in peak detection 3-24...
  • Page 439 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...
  • Page 440 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...
  • Page 441 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...
  • Page 442 Tabs for open files 2-8 in Data Explorer window 2-8 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...
  • Page 443 9-4, 9-5 extracted ion chromatogram created when you right-click-drag to apply custom label 9-15 general 9-3 Isotope calculator 6-19 Troubleshooting (continued) Link View does not work 9-7 Multiple Charge commands dimmed 5-37 overlaid traces 9-6 overview 9-2...
  • Page 444 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...
  • Page 445 X cursors, setting 1-27 x,y data pairs, copying 1-39 in chromatogram header 2-30 see also Extracted absorbance chromatogram (XAC) X-axis chromatogram 2-11 setting range 2-11 spectrum 2-11 see also Extracted ion chromatogram (XIC) in chromatogram header 2-31 y and b ion pairs, labeling C-11 Y cursor, setting 1-27 Y-axis offsetting chromatogram 4-27...
  • Page 446 Index-30 Applied Biosystems...
  • Page 447 Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800 Toll Free (In North America): +1 800.345.5224 Fax: +1 650.638.5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches into 150 countries on six continents.

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