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Applied Biosystems Data Explorer 4 Series, User Manual

Get the most out of your Applied Biosystems Data Explorer 4 Series with our comprehensive User Manual. This indispensable guide covers every aspect of the product, ensuring seamless operation and maximum efficiency. Download this manual for free from our website and revolutionize your data exploration experience.

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Chapter  6    Using Tools and Applications

6-2

Applied Biosystems

6

6.1  Using the Elemental 

Composition Calculator

This section includes:

•

Determining elemental composition

•

Setting limits

6.1.1  Determining Elemental Composition

This section includes:

•

Description

•

Determining identity of fragment masses

•

Procedure

•

Calculating

•

Results

•

If no results are displayed

•

Displaying the theoretical isotope distribution

Description

The Elemental Composition calculator determines possible 
elemental or amino acid compositions for a given mass. The 
application then generates a theoretical isotope pattern using 
the Mass Resolution specified in Basic Peak Detection 
settings, compares each observed mass and isotope pattern 
to the theoretical mass and isotope pattern for each possible 
composition, and reports an isotope match score that reflects 
how closely they match.

Determining

identity of

fragment masses

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. For more information, 
see “Setting spectrum labels” on page 3-56.

Summary of Contents for Data Explorer 4 Series

Page 1: ...Data Explorer Software Version 4 Series Software User Guide ...

Page 2: ...mages in connection with or arising from the use of this document Applied Biosystems is a registered trademark of Applera Corporation or its subsidiaries in the U S and certain other countries 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 t...

Page 3: ...rocessing and Graphic Settings SET 1 17 1 4 3 Customizing Toolbars 1 21 1 5 Setting Graphic Options 1 23 1 5 1 Changing Background Color 1 23 1 5 2 Customizing Graphic Options 1 24 1 5 3 Reverting to Previous Graphic Options 1 29 1 6 Managing Files 1 30 1 6 1 Converting SPC File Format to DAT File Format Mariner Data Only 1 30 1 6 2 Converting Data from Profile to Centroid Mariner Data Only 1 33 1...

Page 4: ...race 2 15 2 4 3 Dividing the Active Trace 2 15 2 4 4 Adding Traces from the Same Data File to a Window 2 16 2 4 5 Removing Traces 2 21 2 4 6 Expanding and Linking Traces 2 21 2 4 7 Recalling and Rearranging Traces Processing History 2 22 2 4 8 Overlaying Traces 2 24 2 4 9 Annotating Traces 2 28 2 4 10 Viewing Trace Labels 2 30 2 4 11 Printing Traces 2 33 2 5 Working with Multiple Data Files 2 36 2...

Page 5: ...ection Parameter Descriptions 3 19 3 2 5 Charge State Determination and Examples 3 32 3 3 Peak List 3 37 3 3 1 Displaying the Peak List 3 37 3 3 2 Inserting Peaks in the Peak List 3 39 3 3 3 Saving the Peak List 3 40 3 3 4 Sorting Filtering and Printing the Peak List 3 42 3 4 Deisotoping a Spectrum 3 45 3 5 Peak Labeling 3 52 3 5 1 Charge State Labels 3 53 3 5 2 Setting Chromatogram and Spectrum P...

Page 6: ... 27 4 7 1 Using Baseline Offset 4 27 4 7 2 Using Baseline Correction 4 29 4 8 Using UV Trace Offset Mariner Data Only 4 30 Chapter 5 Examining Spectrum Data 5 1 Overview 5 2 5 2 Creating a Combined Spectrum 5 4 5 3 Manual Calibration 5 5 5 3 1 Overview of Manual Calibration 5 5 5 3 2 Manually Calibrating 5 7 5 3 3 Creating or Modifying a Calibration Reference File REF 5 17 5 3 4 Reverting to Instr...

Page 7: ...rithmetic 5 64 Chapter 6 Using Tools and Applications 6 1 Using the Elemental Composition Calculator 6 2 6 1 1 Determining Elemental Composition 6 2 6 1 2 Setting Limits 6 7 6 2 Using the Isotope Calculator 6 13 6 3 Using the Mass Resolution Calculator 6 20 6 4 Using the Signal to Noise Ratio Calculator 6 23 6 5 Using the Ion Fragmentation Calculator 6 25 6 6 Using the Elemental Targeting Applicat...

Page 8: ...ks from Complex Digests 7 18 Chapter 8 Viewing Voyager PSD Data 8 1 Displaying PSD Data 8 2 8 2 Applying Fragment Labels 8 8 8 3 Calibrating a PSD Spectrum 8 10 8 3 1 Checking Peak Detection 8 11 8 3 2 Calibrating 8 12 8 3 3 Creating PSD Calibration CAL Files and Applying to Other Data Files 8 20 8 3 4 Creating PSD Calibration Reference REF Files 8 21 8 3 5 Changing the Precursor Mass 8 23 Chapter...

Page 9: ... Appendix C Data Explorer Toolbox Visual Basic Macros C 1 C 1 Overview C 2 C 2 Preparing Data Before Accessing Macros C 3 C 3 Accessing the Macros C 4 C 4 Using the Ladder Sequencing Toolbox C 5 C 5 Using the Peptide Fragmentation Toolbox C 9 C 6 Using the Polymer Analysis Toolbox C 15 C 7 Using MS Fit MS Tag Toolbox C 18 Index ...

Page 10: ...Table of Contents x Applied Biosystems ...

Page 11: ...ystems Data Explorer Software User s Guide is organized into chapters and appendixes Each chapter page is marked with a tab and a header to help you find information The table below describes the material covered in each chapter and appendix Chapter Appendix Content Chapter 1 Data Explorer Basics Describes file formats file management the parts of the Data Explorer window and how to customize the ...

Page 12: ...xamples for Mariner data and Voyager data Examples include how to improve the signal to noise ratio for reserpine deconvolute unresolved peaks in cyctochrome c Mariner data and label partially resolved peaks Voyager data Chapter 8 Viewing Voyager PSD Data Describes how to view label and calibrate PSD data Chapter 9 Troubleshooting Includes symptoms and possible causes of and corrective actions for...

Page 13: ... operator For example NOTE If you are prompted to insert the boot diskette into the drive insert it then press any key A caution provides information to avoid damage to the system or loss of data For example CAUTION Do not touch the lamp This may damage the lamp A warning provides information essential to the safety of the operator For example WARNING CHEMICAL HAZARD Wear appropriate personal prot...

Page 14: ...ion about the Voyager DE Workstation Printer documentation depends on the printer you purchase Use this documentation to set up and service your printer Microsoft Windows NT User s Guide and related documents Use this guide to learn detailed information about the Microsoft Windows NT user interface Send us your comments We welcome your comments and suggestions for improving our manuals You can sen...

Page 15: ...a 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 1 23 1 6 Managing Files 1 30 ...

Page 16: ...ures Data Explorer software includes a suite of tools and processing options to allow you to graphically and interactively manipulate chromatographic and mass spectral data For example you can Smooth and noise filter data Automatically and manually calibrate spectral data Set peak detection parameters and custom labels for regions of the trace Detected peaks can be evaluated for charge state deter...

Page 17: ...ndow opens The Data Explorer window is blank with only a few menus displayed until you open a data file Figure 1 1 shows the Data Explorer main window with a Mariner data file open Figure 1 2 shows the Data Explorer main window with a Voyager data file open To exit the software select Exit from the File menu in the Data Explorer window The Data Explorer software closes Figure 1 1 Data Explorer Win...

Page 18: ...Mariner and Voyager Mariner Black background yellow traces and green labels Voyager White background blue traces and red labels You can customize the default colors as needed See Section 1 5 1 Changing Background Color NOTE For consistency all Mariner and Voyager screen examples in the following sections of this User s Guide are shown with a white background ...

Page 19: ...ta to ASCII format for import into other software applications or import ASCII results For more information see Section 1 6 3 Converting to and Exporting ASCII Data and Section 1 6 4 Importing a Trace in ASCII Format 1 2 2 Data DAT File Format DAT file format Data generated by Mariner and Voyager systems is stored in DAT file format The DAT file format incorporates all information about how a data...

Page 20: ... MSB files in Data Explorer You cannot convert them to DAT format NOTE Voyager SPC format files are not supported in the Data Explorer software Extracting information from DAT files You can also store parameter settings in separate files by extracting information from a DAT file as needed for use with other files For more information see Section 1 6 5 Extracting and Saving Information from DAT RSD...

Page 21: ...y LBC Chromatogram label information See Section 3 5 3 Setting Custom Peak Labels LBS Spectrum label information See Section 3 5 3 Setting Custom Peak Labels Process CAL Calibration constants generated by mass calibration For more information see Exporting BIC MSM and CAL files on page 1 36 and Applying new constants to additional files on page 5 16 CTS Processed trace that you access by selecting...

Page 22: ...Control Panel NOTE Voyager data files in SPC format have a file structure different from Mariner data files in SPC format and are not supported in the Data Explorer software MS Voyager only Data file format for files acquired before Version 5 0 of the Voyager Instrument Control Panel MSA and MSF Voyager only PSD data file format for composite and fragment files acquired before Version 5 0 of the V...

Page 23: ...ol Panel for a snapshot of chromatogram data NOTE Results for a DAT file are stored within the DAT file not as a separate file RST Results file saved from Mariner SPC format data file versions earlier than 3 0 in the Data Explorer software after a spectrum is manually processed See Section 2 8 Saving Opening and Deleting SPC Results Files Mariner Data Only Voyager MS format data file in the Data E...

Page 24: ...om DAT files See Section 2 7 Exporting Opening and Deleting RCD and RSD Results Files Mariner Data Only RSD Spectrum results file exported from DAT files See Section 2 7 Exporting Opening and Deleting RCD and RSD Results Files Mariner Data Only Table 1 2 Additional File Types Continued Category File Type File Content ...

Page 25: ...ion describes Overview Toolbar Chromatogram and Spectrum windows Tabs for data files Data file names Output window Overview Figure 1 3 shows the Data Explorer window with Mariner data Figure 1 3 Parts of the Data Explorer Window Toolbar Chromatogram Spectrum Data file name Tabs for open data files window window Output window ...

Page 26: ... Voyager data Table 1 4 Table 1 3 Mariner Data Displayed in Chromatogram and Spectrum Windows Window Mariner Data 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 Constant Neutral Loss Chromatogram CNL optional Extracts only the response from peaks that ar...

Page 27: ...indow Voyager Data CHRO Window is not displayed by default Optionally displays Total Ion Current TIC for multiple spectra DAT files if you select Restore Chromatogram from the View menu NOTE DAD functions are not supported for Voyager data SPEC Depends on the type of data file you open Single spectrum files Displays the spectrum and labels the plot as spectrum 1 Multiple spectrum files Displays th...

Page 28: ...ectrum related commands are displayed if you select menus when the Spectrum window is active Only chromatogram related commands are displayed if you select menus when the Chromatogram window is active For Voyager multiple spectra DAT files UV functions are disabled Tabs for data files Chromatogram and Spectrum windows display a tab at the bottom see Figure 1 3 on page 1 11 that allow you to switch...

Page 29: ...ner 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 peak detection and integration For more information see Section 3 3 Peak List Spectrum Peak List Displays results of spectrum peak detection integration and centroiding For more information see...

Page 30: ...sition Calculator Elemental Targeting Displays results for the Elemental Targeting application For information see Section 6 6 Using the Elemental Targeting Application Displaying clearing and closing The Output window is automatically displayed when you generate results for example when you calculate resolution To display the Output window manually select Output Window from the View menu To clear...

Page 31: ...ng a value or selecting a button closing the dialog box then closing the data file you are viewing The next time you open the dialog box the last settings specified are displayed 1 4 2 Customizing Processing and Graphic Settings SET This section includes Overview of processing and graphic settings What settings contain Default processing and graphic settings Default graphic settings Customizing se...

Page 32: ...tain Graphic settings include the attributes you set in Graphic Options described in Section 1 5 Setting Graphic Options Processing settings include Peak detection parameters described in Section 3 2 4 Peak Detection Parameter Descriptions Smoothing points described in Section 5 7 Noise Filtering Smoothing Automatic calibration settings described in Section 5 4 Automatic Calibration NOTE You can o...

Page 33: ...se SET files contain the default graphic settings applied when you select White Background or Dark Background after selecting Default from the Display menu For information see Section 1 5 1 Changing Background Color Customizing settings saved in a data file You can customize settings saved in a data file by adjusting graphic or processing settings in the Data Explorer window Settings are saved wit...

Page 34: ...stomize processing and graphic settings as needed For information on the contents of processing and graphic settings see What settings contain on page 1 18 6 To save settings select Settings from the File menu then select one of the following Save Processing Settings As Save Graphic Settings As Save Graphic Processing Settings As 7 In the Save As dialog box type the name of the SET file then click...

Page 35: ...ttings when you open a file or files See Section 2 1 Opening and Closing Data Files 1 4 3 Customizing Toolbars Customizing toolbars To customize the toolbar 1 Select Customize Toolbar from the Tools menu to display the Customize dialog box 2 To display or hide a toolbar section click the Toolbars tab then select or deselect a toolbar 3 To add a button to a toolbar click the Commands tab select the...

Page 36: ...bar within the Data Explorer window by click dragging the double bar at the left side of the toolbar section To move the toolbar section back to the top of the window click drag the toolbar back to the original position You can display or hide each section individually add or remove buttons on the toolbar and rearrange the order of buttons displayed To do so you must have the Customize dialog box ...

Page 37: ...d color by selecting Default from the Display menu then selecting White Background Displays blue traces and red labels by default Default settings are contained in DEFAULTWHITE SET Dark Background Displays yellow traces and green labels by default Default settings are contained in DEFAULTBLACK SET NOTE These SET files contain graphic settings only They do not contain processing settings You can cu...

Page 38: ... compression Accessing graphic options To access graphic options 1 Display the trace of interest 2 From the Display menu select Graphic Options 3 To use the graphic options settings for all traces click Use same settings for all traces in the View Setup tab 4 Click a Graph Setup tab in the Graph and Plot Options dialog box Figure 1 4 5 Set colors line widths data cursors and graphic compression as...

Page 39: ... 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 1 Select Graphic Options from the Display menu 2 Set colors in the Graph Setup tab see Figure 1 4 Line or vertical bar traces Line width Data cursor Graphic compression Peak bounds ...

Page 40: ...aid When you use Auto Color The active trace color stays at its original setting Other trace colors are set based on the active trace color For example if the active trace is yellow other traces are assigned the colors pale blue pale green and medium gray which are the colors listed after yellow in the Trace color list excluding white NOTE White is not used in Auto Color because white may not prin...

Page 41: ...rsors before printing To set the cursor mode select the appropriate label type as described below Label Type Options Y Label Absolute Displays the number of counts BP Relative Displays the Intensity value relative to the base peak in the trace Includes BP marker on the cursor label Display Relative Displays the Intensity value relative to the largest peak in the current display range X X Label Abs...

Page 42: ...omputer monitor For example assume that a data file contains 10 000 data points and needs compression to 1 000 data points to fit on your computer screen Every 10 data points will be compressed into a single data point To set graphic compression settings 1 Select Graphic Options from the Display menu 2 In the Graph Setup tab of the Graph and Plot Options dialog box see Figure 1 4 on page 1 25 sele...

Page 43: ...ic Setting Revert to Last Saved Graphic Processing Settings Reverts to the last graphic and processing settings saved in the data file To access select Settings from the File menu then select Revert to Last Saved Graphic Processing Settings 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...

Page 44: ...rom data files 1 6 1 Converting SPC File Format to DAT File Format Mariner Data Only This section includes When to convert Before you begin Converting Viewing file properties Searching file properties NOTE You cannot convert Voyager MS files to DAT format When to convert You are not required to convert Mariner SPC file format for data acquired in software versions earlier than 3 0 to DAT file form...

Page 45: ... the File menu The Open dialog box is displayed 2 From the Files of type drop down list select All Files A list of all files contained in the directory is displayed 3 Check that the SPC and CGM files are present Converting In the Data Explorer window 1 Open or click the SPC file to convert 2 Select Convert from the File menu 3 Select New Data Format The Convert to DAT Format dialog box is displaye...

Page 46: ... the file of interest 2 In Windows NT Explorer select the file then right click 3 Select Properties from the menu 4 Click the Summary tab NOTE If the Summary tab is not available the file may be open in Data Explorer Close the file and repeat the steps above Searching file properties To search for a file based on file properties 1 In Windows NT Explorer select Find from the Tools menu then select ...

Page 47: ...ore you begin on page 1 31 If the SPC and CGM files are not in the same directory when you open the SPC file a Failed to open chromatogram data message is displayed Converting to centroid To convert from profile to centroid format 1 Open or activate the DAT or SPC file to convert 2 Select Convert from the File menu then select Centroid The Save As dialog box is displayed The software appends a CT ...

Page 48: ... select ASCII text A Save As dialog box is displayed 3 Specify the name and destination for the file to be exported By default the software assigns a TXT extension to the file 4 Click OK Exporting a trace to ASCII format You can export a selected trace to ASCII format for display in Data Explorer software or for use in another application To export trace data to ASCII format 1 With a data file ope...

Page 49: ...he correct window type Importing a Trace in ASCII Format To import an ASCII trace 1 Open a data file and activate a Chromatogram or Spectrum window NOTE You must open a data file before you can import ASCII data even if the data file is unrelated to the imported data 2 Select Add Remove Traces from the File menu then set the Replace Mode by selecting one of the following Replace the Active Trace A...

Page 50: ...RSD spectrum results file and RCD chromatogram results file then save the information as a stand alone file for use with other files Instrument settings BIC MS Method MSM Mariner data only Calibration constants CAL Processing graphic settings SET Spectrum or chromatogram peak labels LBS or LBC ASCII Spectrum and ASCII Chromatogram TXT Exporting BIC MSM and CAL files To export Instrument settings B...

Page 51: ...ile as described above Then export the BIC files from the MSM file using the Export button in the MS Method editor For more information on exporting a BIC file from MSM see the Mariner Workstation User s Guide 3 In the Save As dialog box type a name for the exported file then click Save Saving SET files To save processing and graphic settings SET from a DAT or results file 1 Open or activate the D...

Page 52: ...ight clicking then selecting Copy Trace Data For more information see Section 2 5 2 Copying Traces from Multiple Data Files to a Window Displayed Peaks Copies peak list entries for the peaks displayed in the active window All Peaks Copies peak list entries for all peaks in the active view Mass List Copies all centroid or apex masses from the peak list for the active Spectrum window Copy trace imag...

Page 53: ...3 Paste the data into an appropriate application for example another Data Explorer trace window or Microsoft Excel When pasted into a Data Explorer trace window the trace appears with the filename in parentheses and the trace label from the copied trace CAUTION A trace pasted into an active Data Explorer window contains only the data points for the trace The Sample Info and Instrument settings tab...

Page 54: ...he trace window is copied 3 From the Edit menu select Copy then select Displayed Peaks 4 Paste the data into an appropriate application for example Microsoft Excel Copy all peaks Use this method to copy the peak list for all peaks in the active trace To copy only the section of the peak list pertaining to the peaks displayed in the active trace window see Copy displayed peaks on page 1 39 NOTE Cop...

Page 55: ...ts or other applications without having to delete header information To copy all centroid or apex mass data from the spectrum peak list to the clipboard 1 Select the Spectrum window to copy from 2 Select Peak Label from the Peaks menu 3 Specify the Mass Type by selecting Apex or Centroid For more information see Section 3 5 2 Setting Chromatogram and Spectrum Peak Labels 4 Click OK 5 From the Edit...

Page 56: ...Chapter 1 Data Explorer Basics 1 42 Applied Biosystems 1 ...

Page 57: ...ng Data Files 2 2 2 2 Adjusting the Display Range 2 11 2 3 Organizing Windows 2 13 2 4 Manipulating Traces 2 14 2 5 Working with Multiple Data Files 2 36 2 6 Saving Opening and Deleting DAT Results 2 38 2 7 Exporting Opening and Deleting RCD and RSD Results Files Mariner Data Only 2 39 2 8 Saving Opening and Deleting SPC Results Files Mariner Data Only 2 40 ...

Page 58: ...recently opened file The File menu lists the last several opened files up to a maximum of nine To quickly open one of these files select it from the list NOTE If you access Data Explorer software from the Voyager Instrument Control Panel the most recently saved Voyager data file is displayed in the Data Explorer window This data file and other recently acquired data files are listed in the Data Ex...

Page 59: ... files to open then click Add or Add All Add All is not active if the number of selected files exceeds eight NOTE You can also select files by double clicking the file name in the file list The files are listed in the Files Selected box Displaying acquisition comment 4 To display acquisition comments before opening the data file right click a file name in the top pane of the dialog box select Prop...

Page 60: ...Settings Applies settings from the appropriate default SET file for your system See Default processing and graphic settings on page 1 18 Use Selected Set File Opens the Restore Graphics and Processing Settings dialog box where you select the SET file to open For information on customizing a SET file see Section 1 4 2 Customizing Processing and Graphic Settings SET 6 Click Finish to open the select...

Page 61: ...e User s Guide 2 5 2 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: ... more information see Types of Mariner data on page 4 2 and Creating an Extracted Absorbance Chromatogram XAC Mariner Data Only on page 4 13 Type of Data Spectrum Trace Labels 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 ...

Page 63: ...A Chromatogram window is displayed for the data file NOTE Diode array support and UV display are disabled for Voyager chromatogram traces 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 All functions that ...

Page 64: ... Figure 2 4 to activate the file Figure 2 4 Tabs for Open Data Files If the window for the tab you click is already open clicking the tab activates the window If the window for the tab you click is minimized clicking the tab activates the title bar for the window Double click the active title bar to display the window Using Activate File To move between Spectrum windows of open data files using Ac...

Page 65: ...Files Data Explorer Software User s Guide 2 9 2 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 ...

Page 66: ...File menu to close all files In the Open File dialog box select any open files from the Files Selected list click Remove then click Finish to close the selected files Select Exit from the File menu to close all files and exit the software If you enabled Processing History and selected the Show Save History option a dialog box is displayed Click Save or Purge For more information see Setting Proces...

Page 67: ...lay menu select Range 3 Select X Range to set the x axis range The scaling units depend on the window you are scaling Chromatogram Scales in the same units currently displayed in the Chromatogram window Spectrum Number or Time Mariner data only Spectrum Scales in m z units 4 Select Y Range to scale the y axis range The Y Axis Setup dialog box Figure 2 6 is displayed Figure 2 6 Y Axis Setup Dialog ...

Page 68: ...is select Graphic Options from the Display menu click the Graph 1 Setup tab then deselect the Show Right Y Axis check box Absolute Value Sets the trace to the Y Display Range you enter in the Y Display Range From To boxes Display Min Max Sets the trace display to the minimum and maximum Y values Minimum Absolute Max Y Use Limit Sets the minimum value for Y axis scaling Useful to maintain relative ...

Page 69: ...s 1 Open the data files you want to link 2 In the first data file click the window Chromatogram or Spectrum that you want to link to another data file then select Link View from the View menu NOTE Clicking in the toolbar links traces not views 3 Repeat step 2 if you want to link both windows 4 Repeat step 2 and step 3 for the remaining data files NOTE You must select Link View for each window and ...

Page 70: ...alling and rearranging traces Processing History Overlaying traces Annotating traces Viewing trace labels Printing traces 2 4 1 Zooming Centering and Customizing a Trace Zooming and unzooming You can expand zoom an area of a trace by click dragging a box around the area of interest You can also click buttons in the toolbar to Zoom in NOTE Display data cursors then click the point you want to zoom ...

Page 71: ...ession 2 4 2 Duplicating a Trace To make a duplicate of a trace 1 Click the trace 2 From the Display menu select Duplicate Active Trace The active trace is duplicated and displayed in another trace position 2 4 3 Dividing the Active Trace You can divide the active trace into equal segments with one command This is useful to expand the x axis range to better see spectral and chromatographic feature...

Page 72: ...race represents the range from 15 to 20 NOTE To restore the display to a single trace select Remove Inactive Traces from the Display menu 2 4 4 Adding Traces from the Same Data File to a Window This section describes Overview Setting the Replace mode Adding a trace Overview By default the Data Explorer software displays Chromatogram and Spectrum windows each contain one trace see Figure 1 3 on pag...

Page 73: ... Replace mode to add to or replace the active trace You can add up to seven new traces to a window to allow you to keep original data displayed when you generate new traces Setting the Replace mode To set the Replace mode 1 From the Display menu select Add Remove Traces The Display Trace dialog box is displayed Figure 2 7 Figure 2 7 Display Trace Dialog Box ...

Page 74: ... is replaced when a new trace is generated Hint A toolbar button is available for switching between Replace and Add mode See Customizing toolbars on page 1 21 for information The button is located in the Graph category 3 If desired you can also add traces by selecting the trace to add from the Select Traces to Display section 4 Click OK Adding a trace To add a new trace to a window 1 Click the Chr...

Page 75: ... 2 19 2 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 Original Added trace traces ...

Page 76: ... 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 Figure 2 9 Added Traces Containing Data For a description of trace labels see Viewing Trace Labels on page 2 30 Original Added trace traces ...

Page 77: ...ive traces from the window 1 Click the trace to keep displayed to make it the active trace 2 Select Remove Inactive Traces from the Display menu 2 4 6 Expanding and Linking Traces When you have more than one trace displayed in a window you can Expand traces Click a trace then click in the toolbar to expand the selected trace for closer examination To display all traces click again Link traces Clic...

Page 78: ...completely Recalling or rearranging To recall or rearrange previously displayed traces 1 Click the trace position in which you want to recall or rearrange a trace 2 From the Display menu select Processing History A submenu is displayed listing the last 16 processed traces viewed in the window NOTE Unprocessed traces and theoretical traces generated using commands on the Applications menu are not l...

Page 79: ...d purges the history log when you close the data file Purging a history file does not affect the data contained in the data file It clears the contents of the CTS file that contains the processing history Save processing history Records all processing functions performed Stores the history log in a CTS file and maintains the history log when you close the data file If you save processing history C...

Page 80: ...pying Traces from Multiple Data Files to a Window Hint The copied traces display the original trace label and filename 2 To use settings other than defaults set attributes for the overlay if needed See Setting overlay attributes on page 2 26 3 Click the trace of interest to activate it NOTE Only the active trace in an overlay is affected by processing tools However all traces are affected by zoomi...

Page 81: ...ributes for the overlay if needed See Setting overlay attributes on page 2 26 3 Click the trace of interest to activate it NOTE Only the active trace in an overlay is affected by processing tools However all traces are affected by zooming functions 4 From the Display menu select Overlay Traces Hint A toolbar button is available to toggle between Overlay and Undo Overlay mode See Customizing toolba...

Page 82: ...her trace If you select Spectrum 3 then click to display Spectrum 2 then click to return to Spectrum 3 Spectrum 3 is no longer smoothed Setting overlay attributes To set overlay attributes 1 Display the individual traces to overlay 2 Select Graphic Options from the Display menu The Graph and Plot Options dialog box is displayed NOTE If traces are overlaid when you select Graphic Options you can se...

Page 83: ...s as needed If you selected Use same settings for all traces click Autocolor to allow the software to automatically assign trace colors NOTE The active trace color stays at its original setting Other trace colors are set based on the active trace color For example if the active trace is yellow other traces are assigned the colors pale blue pale green and medium gray which are the colors listed aft...

Page 84: ...cting commands from the Tools or Applications menu For more information see 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 Click the Results tab in the Output window 3 Select the line...

Page 85: ...iew If you zoom on a different region of the trace and the x coordinate moves out of view the annotated text also moves out of view If you annotate overlaid traces only the text associated with the active trace is displayed NOTE To move the text click drag the text to the desired position 3 To customize the appearance of the annotated text see Section 1 5 Setting Graphic Options 4 To delete an ann...

Page 86: ...n BC Baseline corrected BO Baseline offset BP Base Peak mass and intensity CNL Constant neutral loss chromatogram EF Event filtered chromatogram MS Method data only Mass xxx yyy Extracted ion chromatogram for a mass range where xxx is the starting mass and yyy is the ending mass NFX Noise filtered trace where X is the applied Correlation Factor NRX Noise removed trace where X is the number of stan...

Page 87: ...xx 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 Chromatogram Trace Label Description Trace label Spectrum Trace Label Type of Processing or mmm nnn Mariner data only Added accumulated or subtracted spectrum from spectrum mmm to nnn AC Automatically ma...

Page 88: ...moved trace where X is the number of standard deviations of noise removed RSMX does not apply to Voyager PSD data Default smoothing applied where X is the resolution value from peak detection used to calculate the optimum number of smoothing points to apply at every mass point SC Mariner data only Converted to single charge SMX Smoothed where X is the number of smoothing points applied Stitched PS...

Page 89: ...s by selecting Default from the Display menu then selecting White Background NOTE If you previously modified the colors associated with this command as described in Section 1 5 1 Changing Background Color selecting this command may not set a white background and black traces When you manually set colors note the following Selections set to white or line widths set to 0 may not print on certain pri...

Page 90: ...e along with the settings used to obtain the data Print Spectrum or Chromatogram Trace Prints the active trace Print Spectrum or Chromatogram View Prints all traces in the Spectrum or Chromatogram window Print All Views Prints traces for all open data files NOTE If you select Print All Views when more than two data files are open certain printers may not print the data file name To ensure data fil...

Page 91: ...top click Start then select Settings 2 Click Printers 3 Select the printer name in the displayed list 4 Click File then select Document Defaults 5 In the Page Setup tab select Landscape orientation NOTE If you cannot select Landscape orientation you do not have access permission See your administrator Print Setup The Print Setup function allows you to select a printer and set printer options For m...

Page 92: ...the data files as described in Section 2 1 Opening and Closing Data Files 2 Set the Y Scaling mode to Display Relative as described in Section 2 2 Adjusting the Display Range 3 Link the data files by selecting the Spectrum window for each data file then selecting Link View from the View menu You must select Link View for each data file NOTE Clicking in the toolbar links traces not views 4 Organize...

Page 93: ... or right click the trace then select Copy Trace Data 3 Activate the window in which to paste the trace NOTE The Add Replace Trace state as set in the Display Trace dialog box determines whether the copied trace replaces or is added below the active trace See Setting the Replace mode on page 2 17 4 From the Edit menu select Paste then Trace Data or right click the window then select Paste Trace Da...

Page 94: ...ve As 3 Use the default title or enter a name up to 31 characters for the results in the Title text box then click OK Only the results for the active trace are saved NOTE Results are stored within the DAT file not as separate files Title is an identifier you can use to recall the results It is not a file name Opening results for DAT files To open results for DAT files 1 From the File menu select R...

Page 95: ...of the exported file By default the software assigns a RCD extension for a chromatogram or a RSD extension for a spectrum 4 Click OK Opening results for RCD and RSD files To open results from RCD and RSD files 1 Select Open from the File menu The Select Data File to Open dialog box is displayed 2 From the Files of Type drop down list select Result Spectrum Files RS or Result Chromatogram Files RC ...

Page 96: ... Save As 3 Enter a file name for the result file in the File Name text box Spectrum files are automatically named with an RST extension Chromatogram files are automatically named with an RCT extension 4 Click OK NOTE You can also save Mariner RCT and RST files from DAT format data files in the Instrument Control Panel For more information see the Mariner Workstation User s Guide Opening results fo...

Page 97: ...rmation see Section 5 11 AutoSaturation Correction Mariner Data Only 4 Click the Sample Info tab in the Output window to display the following information for the result file Name of the original raw data file from which the result file was generated Processing functions that were performed and saved in the result file Deleting results for SPC files Use Windows NT Explorer to delete RST and RCT re...

Page 98: ...Chapter 2 Using Chromatogram and Spectrum Windows 2 42 Applied Biosystems 2 ...

Page 99: ...g This chapter contains the following sections 3 1 Overview 3 2 3 2 Peak Detection 3 6 3 3 Peak List 3 37 3 4 Deisotoping a Spectrum 3 45 3 5 Peak Labeling 3 52 3 6 Process that Occurs During Peak Detection Centroiding and Integration 3 67 3 7 Default Peak Detection Settings 3 71 ...

Page 100: ...ion based peak detection routine to calculate peak detection values that provide optimum peak detection for most sample types For more information see Section 3 1 2 The Resolution Based Peak Detection Routine For many applications the default peak detection settings and settings calculated by the resolution based peak detection routine provide acceptable peak detection If default settings do not p...

Page 101: ...ion routine applies to spectral data only You can enable or disable the resolution based peak detection routine as described on page 3 14 Process that occurs When enabled the resolution based peak detection routine automatically Divides a trace into detection ranges based on the expected number of data points across a typical mass spectral peak Applies a Filter Width that is equal to the number of...

Page 102: ... Detection Ranges Calculated by Software Based on Number of Data Points Per Peak number of Flight time data points 2 mass resolution Bin size to which the data point corresponds Where For Mariner data Bin size 1 ns Bin size is an instrumental constant Mass resolution is a user defined value Data type dependent defaults are provided but can be overwritten Expected For Voyager data Bin size is varia...

Page 103: ...e software detects the peak using the settings from the detection range within which the entire peak occurs Range 2 in Figure 3 2 If a peak occurs completely within two ranges for example Peak D in Figure 3 2 the software uses the settings from the higher m z detection region Range 4 in Figure 3 2 PSD peak detection for Voyager data For PSD data the mass time correlation is different from that of ...

Page 104: ...w to approach Mariner peak detection For details on peak detection see Section 3 2 3 Setting Peak Detection Parameters Default peak detection values are listed in Section 3 7 Default Peak Detection Settings Strategy When detecting peaks in Mariner data 1 Open the data file and observe the effects of the default peak detection settings If you are analyzing resolved isotopes default settings should ...

Page 105: ... on peaks This threshold is represented as a percentage of the area of the peak with the largest area and is calculated above the local baseline To determine an appropriate threshold display the Spectrum Peak list note the area of the peak with the greatest area and the areas of unwanted peaks and estimate the percentage to enter as the threshold Specifying a Max Peak Area Threshold is particularl...

Page 106: ...solution on a tall resolved peak in the middle of the spectrum If the resolution result differs by more than 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 ...

Page 107: ...ciated with the observed problem Problem Suggested Actions High mass peaks not detected Decrease Mass Resolution setting The default Mass Resolution settings are optimized for masses below 20 000 Da Noise detected as peaks Increase the Max Peak Area Decrease the Resolution Peaks of interest are not detected Decrease the Max Peak Area NOTE Max Peak Area is calculated above the local baseline and ca...

Page 108: ...etection is still not acceptable adjust the remaining peak detection parameters as described in Section 3 2 3 Setting Peak Detection Parameters Partially resolved peaks not detected If peaks represent two compounds and you want both peaks labeled do either of the following Set Max Peak Area to 0 then adjust the Base Peak Intensity until peaks are detected Click the Peak Processing tab then change ...

Page 109: ...ad Section 3 2 1 Strategy for Mariner Peak Detection Section 3 2 2 Strategy for Voyager Peak Detection Setting chromatogram parameters To set chromatogram parameters for data 1 Click the Chromatogram window to activate it 2 Click the trace of interest 3 Click in the toolbar or select Peak Detection from the Peaks menu The Chromatogram Peak Detection dialog box is displayed Figure 3 3 You can click...

Page 110: ...nd set parameters as needed 5 To apply settings to all traces select Use same settings for all traces in view To set parameters independently for all traces in a window deselect Use same settings for all traces in view 6 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 111: ...est 3 Click in the toolbar or select Peak Detection from the Peaks menu The Spectrum Peak Detection Setup dialog box opens with the Basic Settings tab Figure 3 4 displayed NOTE If you applied Advanced Settings to a data file they override the settings on the Basic Settings tab and the Advanced Settings tab is displayed when you select Peak Detection See Setting Advanced Settings spectrum data only...

Page 112: ...tings and skip to step 7 6 If you are detecting mass spectral data select Use Resolution Dependent Settings if you want the software to Automatically determine the number of data points across a peak Divide the trace into different detection ranges based on the resolution Apply a Filter Width and Increment appropriate for each detection range Apply a Minimum Area of 0 and a Minimum Intensity of 0 ...

Page 113: ... value to use for peak detection The default value for the type of data displayed is acceptable for most applications 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 Ele...

Page 114: ...tab is displayed Figure 3 5 Figure 3 5 Spectrum Peak Detection Setup Peak Processing Tab NOTE You can enable the BP Intensity Threshold Cursor and click drag it to adjust the Base Peak Intensity 2 Set parameters as needed then click Apply to accept the parameters and leave the dialog box open or click OK to accept the parameters and close the dialog box For a description of the parameters see Peak...

Page 115: ...Settings 2 Click the Advanced Settings tab in the Spectrum Peak Detection Setup dialog box NOTE If you select Use Resolution Dependent Settings in the Basic Settings tab Basic Settings override Advanced Settings The Advanced Settings tab is accessible but all parameters are dimmed To make Advanced Settings available for editing select Use Advanced Settings on the Basic Settings tab The Advanced Se...

Page 116: ...tings tab 2 Select Use Resolution Dependent Settings 3 Enter the desired Global Thresholds 4 Click Apply or OK The following occurs Detection ranges Filter Width and Filter Increment previously set on the Advanced Settings tab are reset to defaults Minimum Area and Minimum Intensity are set to 0 Global Threshold settings from the Basic Settings tab are applied to all detection ranges and override ...

Page 117: ...Specifies one or more non contiguous m z ranges for peak detection You can set parameters for each range independently You select a range in the Detection Ranges list box by single clicking the range number To add a detection range do one of the following Select an existing range then click This creates a new range with boundaries ranging from the end of the existing range to the end of the trace ...

Page 118: ...0 and adjust the Max Peak Area 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 and signal spikes Hint If you are analyzing digest data set Max ...

Page 119: ...across a peak you can change the trace display from Line to Vertical Bars Each vertical bar represents one data point For more information see Section 1 4 Customizing the Data Explorer Window 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 Drops a vertical...

Page 120: ...e To be detected peaks must be above this threshold and above the Max Peak Area value If you override Global Thresholds by selecting Use Advanced Settings described on page 3 23 the software ignores Global Thresholds and uses the Base Peak Intensity Max Peak Area Minimum Area and Minimum Intensity thresholds set on the Advanced tab described on page 3 28 to detect peaks for the selected detection ...

Page 121: ...described on page 3 28 to detect peaks for the selected detection range Enable BP Intensity Threshold Cursor When enabled allows you to click drag the cursor to set the Base Peak Intensity Threshold Peak Detection Use Resolution Dependent Settings not available for Voyager PSD data When selected the software automatically determines detection ranges and uses an appropriate Filter Width and an Incr...

Page 122: ...z Decrease this value if you are analyzing higher masses or want to label average isotope masses If you set a resolution value of 1 000 or greater The software uses the setting until it reaches the mass at which isotopic resolution is no longer possible then switches to a resolution of 1 000 the resolution that corresponds to isotopic clusters Less than 1 000 The software uses the setting for all ...

Page 123: ... 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 Trace Settings Use same settings for all traces in view Applies settings to all traces in the active window Table 3 2 Basic Settings Tab Parameters Spectrum Data Only Continued Parameter Description ...

Page 124: ...m and Spectrum Peak Labels Valley to Baseline Draws a vertical line from all valleys to a horizontal baseline The level of the horizontal baseline is determined using the minimum peak valley point left or right for each peak See Figure 3 25 on page 3 70 Valley to Valley Forces a baseline through all valley points See Figure 3 25 on page 3 70 Spectrum Parameters Centroid Specifies the percentage of...

Page 125: ... tolerance of 0 05 m z where proton mass equals 1 007276456 Maximum Charge State is 6 for Mariner data and 1 for Voyager data Maximum Isotopes Specifies the maximum number of peaks included in an isotope cluster NOTE Setting this value too low can result in peaks not being included in the appropriate isotope cluster For more information see Max Isotope set too low on page 3 35 Minimum and Maximum ...

Page 126: ...tab are dimmed To make Advanced Settings available for editing select Use Advanced Settings on the Basic Settings tab Table 3 4 Advanced Settings Spectrum Data Only Parameter Description Peak Detection Settings Detection Ranges Specifies the region of the trace defined by x axis lower and upper boundaries to which the settings apply If you select Use Resolution Dependent Settings in the Basic Sett...

Page 127: ...ends at the end of the trace the region of the existing range is split in half between the existing range and the new range Double click an existing range to manually enter lower and upper boundaries Select a range in the dialog box then click drag the X data cursors labels in the trace to set the lower and upper boundaries To delete a range select the range then click To combine all ranges in the...

Page 128: ...ariner data is 11 000 counts spectrum that has not been accumulated or summed and for Voyager data is 66 000 counts NOTE This parameter was previously named Absolute Threshold Minimum Area Specifies the peak area below which peaks are not detected Calculated relative to peak valleys Hint Display the peak list to determine the appropriate area value to enter Noise Threshold Used to determine peak b...

Page 129: ...y set Filter Width set to a number equal to the number of points across the peak To determine the number of points across a peak you can change the trace display from Line to Vertical Bars Each vertical bar represents one data point For more information see Section 1 4 Customizing the Data Explorer Window Increment Number of data points the filter moves across Use 1 for all applications This value...

Page 130: ...spacing is determined by the Max Charge State plus or minus a tolerance value The tolerance is calculated as proton mass charge state 15 where proton mass equals 1 007276456 During charge state determination the software starts with the most intense peak and the peak spacing determined by the Max Charge state then evaluates all detected peaks The software iteratively decreases the Max Charge State...

Page 131: ...set too low the charge state for all peaks may not be correctly determined In this example with the Max Charge State set to 2 only the first and fourth peaks are labeled with charge states Figure 3 8 on page 3 34 This is because the software Checks for peaks at 0 5 m z from each peak charge state 2 evaluation Finds no peaks and checks for peaks at 1 m z from each peak charge state 1 evaluation Fin...

Page 132: ... 3 9 The software labels the first peak but removes the mass and charge state labels from the 559 3 m z peak indicating that it is part of the 558 3 m z isotope cluster Note that turning on monoisotopic peak list filtering does not affect the mass labels on the 558 6 m z or 558 9 m z peaks because these peaks have no calculated charge and are not part of the determined isotope cluster Figure 3 9 M...

Page 133: ...ge State set to 4 the software labels all peaks as charge state 3 Figure 3 10 Figure 3 10 Max Isotope Set Too Low Max Charge State Set Correctly The effect of setting Max Isotope too low is apparent when you turn on Monoisotopic peak list filtering Figure 3 11 shows that the software has grouped the peaks into two clusters and has identified two monoisotopic peaks Figure 3 11 Max Isotope Set Too L...

Page 134: ...se the Minimum Intensity setting to 85 in the example shown in Figure 3 12 the software cannot determine a charge state for the first peak Figure 3 13 This is because The increased threshold suppresses the detection of the 236 6 m z peak When the software evaluates the peak at 235 6 m z it does not find a peak at the appropriate peak spacing for charge state 1 and therefore determines that the pea...

Page 135: ...he 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 Figure 3 14 Peak List in Output Window Click the Peak List tab ...

Page 136: ...rom the Display menu Lower bound spectrum or minutes Upper bound spectrum or minutes Peak height calculated relative to zero Peak area calculated relative to local baseline Spectrum peak list Each entry represents one spectral peak in the trace and includes Index a sequential number assigned to each entry in the peak list Centroid or Apex mass reflects the Mass Type selected in the Peak Label dial...

Page 137: ...romatogram or spectrum trace of interest 2 From the Peaks menu select Insert Peaks The Insert Peaks dialog box is displayed Figure 3 15 Figure 3 15 Insert Peaks Dialog Box 3 To enter the Left Edge and Right Edge of the peak to insert right click drag over the region of the trace to calculate or type in masses 4 Click Calculate The peak is inserted in the peak list displayed in the Peak list tab in...

Page 138: ... PKT file You can save the contents of the chromatogram and spectrum peak lists as stand alone peak list files PKT Stand alone peak list files can be used in other applications such as Microsoft Notepad Editor or Microsoft Excel To save a peak list as a stand alone PKT file for use in other applications 1 Click the trace of interest 2 Display the peak list of interest in the Output window by click...

Page 139: ...box is displayed 2 Select All Files from the Files of Type drop down list at the bottom of the dialog box Select the directory and file to import then click Open The Text Import wizard is displayed 3 Follow the prompts from the Text Import wizard accepting the default settings provided The peak list is converted to an Excel spreadsheet 4 Select Save As from the File menu The Save As dialog box is ...

Page 140: ...igure 3 16 Sorting the Peak List Filtering the spectrum peak list Peak list filtering allows you to display only desired peaks in the spectrum peak list Only peaks that are included in the peak list are labeled on the trace To filter the peak list 1 From the Peaks menu select Filter Peak List Hint You can also display the Mass Peak List Filter dialog box by right clicking the Spec Peak List then s...

Page 141: ...isotope clusters where the approximate elemental composition and isotopic ratios are not known for example in PSD analysis where the precursor ion filtering enhances the precursor ion isotope intensity relative to the rest of the cluster or in a spectrum that contains molecules with very different elemental compositions For most applications especially peptide analysis the deisotoping function yie...

Page 142: ...ick the peak list then select Print Deleting items from the peak list To delete an item from the peak list select the item right click the spectrum peak list then select Delete Peak The entry is removed from the list and the peak label is removed from the trace To display the deleted item in the peak list again select Peak Detection from the Peaks menu then click OK ...

Page 143: ...Peak deisotoping is an advanced peak filtering method that can determine the relative abundance of multiple components with overlapping isotope distributions The deisotoping algorithm uses the elemental composition that you specify to improve the determination of the monoisotopic mass by considering the centroid masses of all peaks in the isotopic envelope During peak deisotoping For each detected...

Page 144: ...ion is represented in the trace as a centroid bar with its original amplitude Figure 3 17 illustrates how peaks that are and peaks that are not part of an isotope cluster are represented in a deisotoped trace When to use For most applications particularly peptide analysis the deisotoping function yields more useful results than monoisotopic peak filtering described in Section 3 3 4 Sorting Filteri...

Page 145: ...you use the Deisotope function on multiply charged peaks invalid results are reported If first three peaks are part of the same isotope cluster If first three peaks are part of the same isotope cluster but Contribution to expected isotope ratio contaminant is also present If two isotope clusters are present Increased amplitude of first peak indicates it is a monoisotopic peak Increased amplitude o...

Page 146: ...a to 0 before deisotoping to ensure that all peaks of interest are detected For more information see Section 7 2 3 Detecting Peaks from Complex Digests 3 If multiply charged peaks are present convert to a singly charged spectrum See Section 5 10 Converting to a Singly Charged Spectrum Mariner Data Only 4 Select Duplicate Active Trace from the Display menu to keep the original data displayed after ...

Page 147: ...bar is proportional to the peak areas of all peaks in the isotope cluster Troubleshooting If you see several small peaks around a very large peak after deisotoping the software has successfully identified a monoisotopic peak but the isotope cluster does not exactly match the theoretical elemental composition that you specified This may be caused by the following The signal to noise ratio of the sp...

Page 148: ... isotope pattern with four detected peaks Figure 3 19 Spectrum Before Deisotoping After deisotoping Figure 3 20 the trace includes two labeled centroid bars that represent monoisotopic masses indicating that the original trace represents two isotopic envelopes The intensity of a centroid bar is proportional to the area of all peaks in the isotope cluster Figure 3 20 Spectrum After Deisotoping ...

Page 149: ...l spectrum If the original spectrum was an unprocessed spectrum select Spectrum Number from the Display menu The number of the original spectrum is displayed in the Select Spectrum dialog box Click OK If the original trace was a processed spectrum select Processing History from the Display menu then select the original trace ...

Page 150: ... detection parameters described in Section 3 6 Process that Occurs During Peak Detection Centroiding and Integration Charge state determination described in Section 3 2 5 Charge State Determination and Examples Peak list filtering described in Filtering the spectrum peak list on page 3 42 NOTE If peak labeling is disabled no labels are displayed even for peaks in the peak list If you delete a peak...

Page 151: ...ination parameters Max Charge State Max Isotope Min and Max Intensity For a description of these parameters see Advanced Settings spectrum data only on page 3 28 and Charge State Determination and Examples on page 3 32 NOTE If Charge State Determination parameters are set incorrectly the charge state is determined incorrectly For more information see Charge state parameter examples on page 3 32 an...

Page 152: ...rum Peak Labels This section includes Customizing colors font or size Setting chromatogram labels Setting spectrum labels Deleting labels Labels not displayed Charge state not displayed Customizing colors font and size To set colors font and size for peak labels see Section 1 5 Setting Graphic Options Setting chromatogram labels To label chromatogram peaks 1 Click the Chromatogram window to activa...

Page 153: ...l Attributes Overlapping Allows labels to be displayed when peaks are close together Peak bounds Displays peak start peak end and baseline Orientation Specifies Horizontal 45 degree or Vertical labels 6 Select the label content to display for example Spectrum Time Vial Number 7 To create custom labels select User Labels then click User Label Setup See Section 3 5 3 Setting Custom Peak Labels ...

Page 154: ...ia are labeled Setting spectrum labels To label spectrum peaks 1 Click the Spectrum window to activate it 2 From the Peaks menu select Peak Label The Spectrum Peak Label dialog box is displayed Figure 3 22 Figure 3 22 Spectrum Peak Label Dialog Box 3 Select Enable Labeling to label peaks in m z format 4 Set the number of decimal points to be displayed ...

Page 155: ...reviously called Mass Offset Shifts all peak labels by the value entered either positive or negative and can be used to calculate mass differences from a reference peak Right click drag across the reference peak to enter the negative of the value of the peak in the Mass difference text box then click OK The reference peak is labeled with zero and all the other labels are plus or minus their mass d...

Page 156: ...e of peak displayed with a z label where the z represents a positive or negative charge NOTE A z is displayed for positive or negative ions To determine the actual charge of the ions display the Instrument Setting tab in the Data Explorer Output window See Output window on page 1 15 for information Check the listed polarity For spectra acquired in positive ion mode positive ions are produced For s...

Page 157: ...ndow select the item right click the spectrum peak list then select Delete Peak Labels not displayed If a label is not displayed or is displayed incorrectly possible causes are The label is too long peaks are too close together or a peak is very close to the right axis If there is not enough room for the label to be displayed the label is suppressed Zooming the region of interest expands the trace...

Page 158: ...mum charge state for charge state determination is set lower than the charge state of the peak Charge state determination parameters are set such that peaks are determined to have no charge See Section 3 2 5 Charge State Determination and Examples Peaks are not from the same isotope species NOTE If you set Base Peak Intensity or Max Peak Area too low described on page 3 20 noise peaks can be detec...

Page 159: ...is displayed instead of the mass value Customizing colors font and size To set colors font and size for custom labels or peak labels see Section 1 5 Setting Graphic Options Creating custom peak labels To create custom peak labels 1 Click the Chromatogram or Spectrum window to activate it 2 From the Peaks menu select Peak Label The Peak Label dialog box is displayed see Figure 3 22 on page 3 56 3 C...

Page 160: ...nt labeled peak of lower m z 5 Select Match Charge State spectra only if you want the charge state of a peak evaluated before applying the user label When this function is enabled a peak must occur within the specified Mass Tolerance described below and have the specified charge state before the user label is applied Hint The Match Charge State function allows you to screen out peaks that are with...

Page 161: ...ay Peak Mass if Label Type selected is Mass Mass of the peak to which the label applies Mass Difference if Label Type selected is Difference Difference in mass that must exist between peaks to apply user labels Mass Tolerance Mass tolerance that the peak must occur within to apply user labels NOTE If a peak meets the criteria for more than one user label multiple user labels are applied to the pea...

Page 162: ...Setup dialog box The User Label Entry dialog box for the label opens Enter the new values then click OK Hint You can sort the label list by any field by clicking the column header button see Figure 3 23 on page 3 62 10 To save the label settings as a LBC or LBS file click Save As type a file name then click Save 11 Click OK The Peak Label dialog box reappears Chromatogram Parameter Description Lab...

Page 163: ...you make to user labels imported into a DAT file are not saved in the LBC or LBS file from which they originated Click Save As then resave the file if you want the changes saved to the originating file Displaying user labels Select both Enable Labeling and User Labels in the Peak Label dialog box to display user labels Peaks that do not meet the peak detection criteria are not labeled NOTE If you ...

Page 164: ...the label may be suppressed Zooming the region of interest expands the trace and may allow the labels to be displayed To display labels when peaks are close together select Allow overlapping peak labels or change Orientation to Vertical or 45 degrees in the Peak Label dialog box Peak filtering is enabled and only peaks that meet the peak list filtering criteria are labeled Filter Width is set too ...

Page 165: ...eak Detection Routine Starts scanning the chromatogram or spectrum at the origin of the x axis Applies a Gaussian sliding filter to the number of raw data points defined by the Filter Width setting The filter moves across the raw data by the number of data points specified by the Increment parameter An Increment of 1 is used for chromatogram data Searches for an upward to downward inflection point...

Page 166: ...rmine the peak bounds For chromatogram data determines the peak boundaries by Determining the Noise Threshold by performing an automatic signal to noise calculation on the tallest peak in the chromatogram and using 75 percent of the noise value determined as the threshold Scanning from the valley regions toward the apex region using the number of data points defined by the Filter Width If the diff...

Page 167: ...e peak centroid for spectral data by Drawing a projected centroid baseline at the percentage of the peak height specified by the Centroiding entered Centroiding is measured between the top of the peak and 0 For example a centroiding of 10 uses the top ten percent of the peak where peak height is determined from 0 Searching to the left and right of the apex for data points that bound the projected ...

Page 168: ...ds to determine the peak baseline Figure 3 25 Valley to baseline Drops a vertical line from all valleys to a horizontal baseline The level of the horizontal baseline is determined using the minimum peak valley point left or right for each peak Valley to valley Forces a baseline to all valley points Figure 3 25 Valley to Baseline and Valley to Valley Integration Valley to Baseline Valley to Valley ...

Page 169: ...peak detection settings are contained in the following SET files MARINER SET VOYAGERLINEAR SET VOYAGERREFLECTOR SET VOYAGERPSD SET The following table lists the default settings in SET files provided for chromatograms Parameter Chromatogram Settings in All SET Files Mariner and Voyager Basic Settings chromatogram data Detection Range Full Base Peak Intensity 0 25 Max Peak Area 0 25 Filter Width 3 ...

Page 170: ...EAR SET Reflector VOYAGER REFLECTOR SET PSD VOYAGER PSD SET Basic Settings spectrum data Base Peak Intensity 0 25 0 0 0 Max Peak Area 0 25 2 2 2 Use Resolution Dependent Settings ON ON ON OFF Mass Resolution 5 000 2 000 10 000 N A Peak Processing Integration Baseline Setting V to V V to B V to B V to B Centroid 50 50 50 50 Max Charge State 6 1 1 1 Max Isotopes 6 5 10 5 Min Intensity 5 10 10 10 Max...

Page 171: ... can be used for Advanced Detection Range Resolution dependent Resolution dependent Resolution dependent Five ranges Filter Width Resolution dependent Resolution dependent Resolution dependent Range dependent Increment Resolution dependent 1 1 1 Noise Threshold 0 0 0 0 Base Peak Intensity 0 25 0 0 0 Max Peak Area 0 25 2 2 2 Min Intensity 0 0 0 0 Min Area 0 0 0 Range dependent Parameter Mariner Spe...

Page 172: ...Chapter 3 Peak Detection and Labeling 3 74 Applied Biosystems 3 ...

Page 173: ...ns 4 1 Overview 4 2 4 2 Creating an Extracted Ion Chromatogram 4 5 4 3 Creating an Extracted XAC Chromatogram Mariner Data Only 4 13 4 4 Noise Filtering Smoothing 4 17 4 5 Adding and Subtracting Spectra 4 20 4 6 Displaying MS Method Data Mariner Data Only 4 23 4 7 Adjusting the Baseline 4 27 4 8 Using UV Trace Offset 4 30 ...

Page 174: ...es only the base peak in each spectrum Each data point represents the single most intense ion in the corresponding spectrum Analog Trace of the input from an outside source representing any signal that changes over time for example the UV signal from an LC system or the Analyzer Temperature Air Temperature Spray Tip Potential or Nozzle Temperature from the Mariner mass spectrometer DAD TAC Diode A...

Page 175: ...cted Ion Chromatogram XIC which includes only the signal response from a mass window or range For more information see Section 4 2 1 Creating an Extracted Ion Chromatogram XIC Extracted Ion Neutral Loss Constant Neutral Loss chromatogram which extracts only the response from peaks that are separated by a selected mass difference For more information see Section 4 2 2 Creating a Constant Neutral Lo...

Page 176: ...If you have Trace Replace mode set to Replace the new trace replaces the original trace For information on Replace mode see Section 2 4 4 Adding Traces from the Same Data File to a Window To redisplay the original trace select the trace type from the Display menu or click the corresponding toolbar button Select To display Center Window or Range XIC Extracted Ion chromatogram which includes only th...

Page 177: ...differences in a data file correspond to loss of specific fragments by generating a Neutral Loss Chromatogram 4 2 1 Creating an Extracted Ion Chromatogram XIC This section describes how to create an XIC From the Chromatogram window From the Spectrum window From the Chromatogram window To create an extracted ion chromatogram XIC from the Chromatogram window 1 Click the Chromatogram window to activa...

Page 178: ...ow for masses to include NOTE When analyzing multiple components with similar masses set a Window of less than 0 5 to include only the mass of interest Range then type the From and To values for masses to include NOTE To improve the signal to noise ratio in the extracted ion chromatogram set the starting range above the low mass solvent ions NOTE For more information on Constant Neutral Loss chrom...

Page 179: ...lative Creates one extracted ion chromatogram for all masses entered and sums intensities Individual Creates one extracted ion chromatogram for each mass entered 6 Click OK The extracted ion chromatogram is displayed in the Chromatogram window Figure 4 2 on page 4 8 with an XIC trace label and the center mass and window or the mass range indicated in the trace label ...

Page 180: ...r processing 3 In the Spectrum window right click drag over the mass region of interest in the extracted ion chromatogram The width of the box you draw defines the precise mass range used in the extracted ion chromatogram The extracted ion chromatogram is displayed in the Chromatogram window Figure 4 2 with the mass range indicated in the trace label Figure 4 2 Extracted Ion Chromatogram 4 To retu...

Page 181: ... each mass in the peak list to every other mass in the peak list and derives mass differences Takes the total signal of any pairs of peaks that generated the mass difference and includes it in the chromatogram if any mass difference corresponds to a mass difference plus tolerance specified Applications You can generate a CNL extracted chromatogram To determine if a fragment of known mass is presen...

Page 182: ... the Spectrum window right click drag across the peak from which you are measuring the difference This enters a negative value that corresponds to the reference peak in the text box 5 Click OK The reference peak is labeled with zero and all the other peak labels are plus or minus their mass difference from the reference peak For more information on peak labels see Section 3 5 Peak Labeling Procedu...

Page 183: ...to select the mass difference Figure 4 3 Extracted Ion Chromatogram Dialog Box with Neutral Loss Selected 6 Specify the Extraction Mode Accumulative Creates one extracted ion chromatogram for all mass differences entered and sums intensities Individual Creates one extracted ion chromatogram for each mass difference entered 7 Click OK The CNL is displayed in the Chromatogram window Figure 4 5 ...

Page 184: ...ds you can generate a CNL extracted chromatogram Figure 4 4 TIC for Flavonoid Mixture Containing Three Peaks Figure 4 5 shows a CNL extracted chromatogram generated for mass difference of 308 146 m z which corresponds to the diglycosyl group Figure 4 5 CNL Containing Two Peaks The CNL extracted chromatogram contains only two peaks indicating that the diglycosyl group has fragmented from the parent...

Page 185: ...gth window or range 1 Click the Chromatogram window to activate it 2 Display a TAC or Channel chromatogram by selecting Traces from the View menu then selecting the chromatogram 3 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 4 Select Extracted Absorbance from the Process menu or click in the toolbar 5 In the Extracted DAD Chromatogram dia...

Page 186: ...gram 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: ...ive Trace from the Display menu to keep the original data displayed after processing 4 Double click the left mouse button or right click drag over an area of interest in the Chromatogram window A DAD Spectrum appears in the Spectrum window 5 Click the Spectrum window to activate it 6 In the Spectrum window right click drag over the region of interest The width of the box you draw defines the preci...

Page 188: ...Chapter 4 Examining Chromatogram Data 4 16 Applied Biosystems 4 Figure 4 7 Extracted Absorbance Chromatogram 7 To return to the original trace see Returning to the original trace on page 4 4 ...

Page 189: ...ommand 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 Figure 4 8 Noise Filter Smooth Dialog Box ...

Page 190: ...her degree of noise reduction If applying the Noise Filter with a certain Correlation Factor does not yield the necessary noise removal return to the original trace see Returning to the original trace on page 4 4 and apply the Noise Filter again with a higher Correlation Factor setting Applying the filter more than one time with the same Correlation Factor setting does not improve noise removal Hi...

Page 191: ...s the specified number of standard deviations of noise This method slightly affects peak intensity and removes peaks with a signal to noise ratio less than the specified standard deviation In general If you set White Noise Std Dev to removed is 1 68 2 95 3 99 If applying Noise Removal with a certain Std Dev does not yield the necessary noise removal return to the original trace see Returning to th...

Page 192: ...t instrument calibrations use Trace Arithmetic For more information see Section 5 12 Adding and Subtracting Raw or Processed Spectra from the Same or Different Data Files Dual Spectral Trace Arithmetic Adding and subtracting spectra To add and subtract spectra 1 Click the Spectrum window to activate it 2 Select Duplicate Active Trace from the Display menu to keep the original data displayed after ...

Page 193: ...ndow The numbers of the selected spectra are added to the list window A range of spectrum numbers is indicated as X X For example 10 20 indicates spectrum number 10 through spectrum number 20 Click type the spectrum number range to add for example 10 20 then press Enter Hint To sum non contiguous regions of spectra repeat step 4 For example you can add spectra 10 to 20 and spectra 30 to 40 to the ...

Page 194: ...ist are summed before the addition or subtraction occurs NOTE If you select Accumulate mode include at least the same number of spectra in the Subtract range that you include in the Add range For example if you include spectra 775 to 794 in the Add range make sure to include 20 or more spectra in the Subtract range 8 Click OK The subtracted baseline trace is displayed with the added and subtracted...

Page 195: ... Nozzle Potential and one specifying a higher Nozzle Potential to induce in source fragmentation You also assigned a Tag 1 event tag to the first event and the In Source CID event tag to the second event You can filter the TIC or any chromatogram data to display separate traces for the different Nozzle Potentials Displaying acquisition conditions and event tags To display instrument settings MS Me...

Page 196: ...ntaining the event tags 2 Click the Chromatogram window to activate it 3 From the Display menu select Traces then select the trace type to filter 4 If you have more than one trace displayed select the trace to filter 5 From the Process menu select Event Tag Filtering NOTE If the Chromatogram window is not active Event Tag Filtering is not displayed on the Process menu If the active chromatogram wa...

Page 197: ... in an event filtered trace are numbered contiguously 1 2 3 regardless of their relation to the overall acquisition However because the axes of the trace reflect the numbering of the overall experiment you may see spectra with numbers that do not correspond to the x axis Hint Line mode is useful when displaying filtered LC TIC traces Vertical bar mode may be more useful when filtering direct infus...

Page 198: ...spectra from event 1 are included in the filtered trace but not spectra from event 2 A filtered trace displays data points for the selected tags only However if you double click the filtered trace in an area that does not appear to contain data a spectrum is displayed in the Spectrum window Any actions you perform on a filtered trace such as summing include only the spectra displayed in the filter...

Page 199: ...the window on which you want to perform the offset NOTE You can select a Chromatogram or Spectrum window If you do not activate the correct type of window before performing the next step the software does not select values when you click drag on the trace For example you must select a Chromatogram window before starting baseline offset on a chromatogram 2 Select Duplicate Active Trace from the Dis...

Page 200: ...to offset The selected value is displayed in the Right Baseline field 6 To limit the baseline offset to the area between the two selected points select Only Apply from L to R Baseline Midpoint To perform the baseline offset on the entire x axis deselect Only Apply from L to R Baseline Midpoint 7 Click OK The offset baseline trace is displayed with a BO trace label 8 To return to the original trace...

Page 201: ...en out noise peaks For best results apply Baseline Correction then re detect peaks If you use area based thresholding Max Peak Area during peak detection Baseline Correction is not typically needed Max Peak Area compensates for a rising or falling baseline With a baseline that is not at 0 to improve peak detection of small peaks Correcting the baseline To correct the baseline 1 Display the spectru...

Page 202: ...ode on page 2 17 4 From the Process menu select Realign UV Trace The UV Trace Offset dialog box is displayed Figure 4 13 Figure 4 13 UV Trace Offset Dialog Box 5 Enter the retention times as follows Click the Original UV Peak Value min text box then right click drag over the peak to offset in the UV trace Select the Aligned UV Peak To min text box then right click drag over the peak to align with ...

Page 203: ...de 4 31 4 6 Click OK The UV trace peak is shifted to align with the chromatogram trace peak NOTE To restore the original UV trace open the UV Trace Offset dialog box see step 4 then click Reset 7 To return to the original trace see Returning to the original trace on page 4 4 ...

Page 204: ...Chapter 4 Examining Chromatogram Data 4 32 Applied Biosystems 4 ...

Page 205: ...ation 5 26 5 5 Centroiding 5 36 5 6 Mass Deconvolution Mariner Data Only 5 37 5 7 Noise Filtering Smoothing 5 42 5 8 Adjusting the Baseline 5 45 5 9 Truncating a Spectrum 5 56 5 10 Converting to a Singly Charged Spectrum Mariner Data Only 5 59 5 11 AutoSaturation Correction Mariner Data Only 5 62 5 12 Adding and Subtracting Raw or Processed Spectra From the Same or Different Data Files Dual Spectr...

Page 206: ...r single spectrum data files DAD spectrum Mariner DAD data only Click the Chromatogram window select Traces from the Display menu select the DAD TAC or channel of interest then double click any point in the TAC or channel data trace to display the corresponding DAD spectrum Voyager PSD spectra Voyager PSD data files contain the precursor spectrum and fragment ion spectra You can view up to eight s...

Page 207: ...ation on Replace mode see Section 2 4 4 Adding Traces from the Same Data File to a Window To return to the original spectrum If the original spectrum was an unprocessed spectrum select Spectrum from the Display menu The number of the original spectrum is displayed in the Select Spectrum dialog box Click OK If the original trace was a processed spectrum select Processing History from the Display me...

Page 208: ...e a combined spectrum 1 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 2 In the Chromatogram window right click drag across the region of the chromatogram that contains the spectra to combine The combined spectrum Figure 5 1 is displayed Figure 5 1 Combined Spectrum 3 To return to the original trace see Returning to the original spectrum on...

Page 209: ...d for Mariner DAD data 5 3 1 Overview of Manual Calibration Overview During manual calibration You specify a calibration reference file REF that contains reference masses The software matches peaks by comparing observed masses in the spectrum to reference masses The software lists masses that match within the specified peak matching criteria and calculates calibration constants You can apply the c...

Page 210: ... these files as a starting point and add delete reference masses as needed For information see Section 5 3 3 Creating or Modifying a Calibration Reference File REF When to use manual calibration Use manual calibration when you Calibrate Voyager data Calibrate Mariner data and any of the following occur You have one or only a few spectra to calibrate You do not know in advance which reference masse...

Page 211: ...m 0 not from the local baseline For information see Section 5 8 2 Using Baseline Correction Noise filter or use default smoothing Use the method appropriate for your data to remove noise spikes For more information see Section 5 7 Noise Filtering Smoothing It is critical to perform both these functions which affect the peak centroid before calibration It is also good practice to perform the same p...

Page 212: ...le spectrum 1 Click the Spectrum window to activate it then select the spectrum trace of interest 2 From the Peaks menu select Peak Label set the Mass Label Type to Centroid then click OK NOTE For spectra containing broad peaks that have unresolved adducts or impurities such as proteins you may obtain better results if you use apex instead of centroid settings 3 From the Process menu select Mass C...

Page 213: ...ction 5 3 3 Creating or Modifying a Calibration Reference File REF 5 Enter reference matching and calibration criteria Minimum Intensity Peaks must be above this intensity to be considered a match Select the unit for Minimum Intensity Relative Intensity or Absolute Counts Mass Tolerance Peaks must be within this tolerance of the theoretical m z to be considered a match Select the unit m z or ppm ...

Page 214: ...han broader peaks Height More intense peaks are weighted more than less intense peaks Manually matching peaks 7 To manually select the reference mass for a peak right click drag over the peak of interest The Select or Create Reference Peak Information dialog box Figure 5 3 is displayed and lists all masses in the selected reference file The selected entry is the nearest match in the calibration re...

Page 215: ...formation in the appropriate text boxes click Save or Save As to add the information to the reference file then click OK to accept the reference mass for matching Type new reference mass information in the appropriate text boxes click Update to replace an entry in the reference file click Save or Save As to add the information to the reference file then click OK to accept the reference mass for ma...

Page 216: ...ched list then click Delete Selected Match Click Eliminate Fit Outlier The software deletes from the calibration the data point with the largest calibrated Fit Error difference between the calibrated mass and the reference mass as reported in the Output window Figure 5 4 NOTE The point deleted may not be the point with the largest Initial Error difference between the pre calibration observed mass ...

Page 217: ...layed spectrum click Plot The spectrum is calibrated and displayed with an MC trace label The calibration statistics are displayed in the Result tab of the Output window Figure 5 5 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 The 267 Da mass is removed when you it generated the largest fit e...

Page 218: ...statistics are listed at the top of the list Automatically matching peaks You can automate peak matching by clicking Match Peaks instead of right click dragging individual peaks and selecting the mass If any masses within the tolerances of any of the masses listed in the calibration reference file are found in the spectrum the matches are displayed in the Calibration Mass Peak Selection window wit...

Page 219: ...Each spectrum in the data file is calibrated when displayed Voyager data Apply Calibration The current spectrum is calibrated and displayed with an MC trace label The calibration constants are saved with the spectrum Apply to All NOTE This button is displayed only if you are calibrating a Voyager multispectrum data file All spectra in the data file are calibrated using the currently displayed cali...

Page 220: ... from the calibration in the data file They are calculated from the listed masses Export a calibration from a previously calibrated data file Open an existing data file then export the calibration constants in a CAL file See Exporting BIC MSM and CAL files on page 1 36 Applying new constants to additional files You can apply calibration constants from a CAL file to any data file To apply the new c...

Page 221: ...thod data Apply Calibration All spectra in the data file are calibrated and displayed with an MC trace label The calibration constants are saved with the data file Each spectrum in the data file is calibrated when displayed Voyager data Apply Calibration The current spectrum is calibrated and displayed with an MC trace label The calibration constants are saved with the spectrum Apply to All NOTE T...

Page 222: ...ss Charge defaults to 0 if you do not specify Type Resolved Average or Unknown defaults to Resolved if you do not specify Name optional Elemental composition optional NOTE If you view the reference file in a text editor such as Notepad Editor the order of the information listed in the reference file is different than the order of the information displayed in the Reference Mass dialog box see Figur...

Page 223: ... Type the Name and Theoretical m z for a reference compound then select the charge state Optionally enter the Elemental Composition for the compound 3 Specify the mass type Resolved Isotope Mass or Average Mass If you are adding masses for highly charged compounds see Specifying mass type for highly charged narrow peaks on page 5 21 4 Click Insert 5 Repeat step 2 through step 4 for remaining compo...

Page 224: ...ion and type a name for the file then click OK The name of the reference file used for mass calibration is stored in the DAT file and is displayed in the Calibration Reference File field when you open the Manual Calibration dialog box again NOTE Only the name and path of the reference file are stored in the DAT file Contents of the file are not stored Modifying a calibration reference file Hint Yo...

Page 225: ...s in the list is considered during calibration If the mass list contains duplicate entries the calibration may return an invalid number of matches 8 Click Save or Save As 9 In the Save As dialog box select a location and type a name for the file then click Save Specifying mass type for highly charged narrow peaks When specifying highly charged non isotopically resolved species with peaks less than...

Page 226: ... to Instrument Calibration The spectrum is recalibrated with the original calibration constants used during acquisition Mariner data or default calibration Voyager data and displayed with an MC trace label The original and new constants are displayed in the Output window 2 To save the reverted calibration with the data file select Mass Calibration from the Process menu then If you are calibrating ...

Page 227: ...ce label The calibration constants are saved with the spectrum Apply to All NOTE This button is displayed only if you are calibrating a Voyager multispectrum data file All spectra in the data file are calibrated using the currently displayed calibration and are displayed with an MC trace label The calibration constants are saved with the data file Each spectrum in the data file is calibrated when ...

Page 228: ... Instrument Control Panel to compensate for the altered parameters masses in the data file may be significantly different from the reference masses For information on calibrating in the Instrument Control Panel see the Mariner Workstation User s Guide Before performing automatic matching set the Mass Tolerance and Minimum Intensity appropriately to match the expected reference masses After automat...

Page 229: ...meters Polarity Instrument mode If you import a calibration that was generated using settings that are different from the current Polarity and Instrument Mode settings an error message is displayed NOTE The calibration of the mass scale changes when you change the Accelerating Voltage Grid Voltage or Delay Time Default calibration adjusts for these changes However you observe more accurate calibra...

Page 230: ...utomatic calibration for Voyager data During automatic calibration During automatic calibration You specify auto calibration settings reference masses matching criteria and fit rejection parameters Auto calibration settings are saved as part of processing settings in a DAT file If Auto Calibrate is enabled in the Data Explorer software Mariner data only or if the Automatic Calibration function is ...

Page 231: ...for Mariner data The Automatic Calibration function in Data Explorer is useful for quickly calibrating all spectra in a Mariner data file after you prepare automatic calibration settings You use the Automatic Calibration function in Data Explorer to prepare Automatic Calibration settings contain reference masses and other matching information and to calibrate the data You can also use the Automati...

Page 232: ...alibrate data as it is acquired You use the Automatic Calibration function in Data Explorer to prepare Automatic Calibration settings contain reference masses and other matching information You then save a SET file that contains the settings then specify the SET file in the Sequence Control Panel The Sequence Control Panel automatically calibrates spectra using the calibration settings contained i...

Page 233: ...T information from this file by selecting Settings from the File menu then selecting Save Processing Settings As For more information see Section 1 6 5 Extracting and Saving Information from DAT RSD and RCD Files 3 Open or activate the new file 4 Copy the settings to the new file by selecting Settings from the File menu then selecting Restore Processing Settings For more information see Section 1 ...

Page 234: ...tain better results if you use apex instead of centroid settings 3 From the Process menu select Mass Calibration then select Automatic Calibration The Automatic Calibration Settings dialog box is displayed Figure 5 7 Figure 5 7 Automatic Calibration Settings Dialog Box 4 Click Select New File then select a calibration reference file For information on creating a reference file see Section 5 3 3 Cr...

Page 235: ...ist to individually select reference masses to add NOTE If the current list already contains 10 reference masses you must delete a mass before you click Add References to List If you click Add Reference to List the Select or Create Reference Peak Information dialog box Figure 5 8 is displayed and lists all masses in the reference file If you click Add All this dialog box is not displayed Figure 5 ...

Page 236: ...e information see Modifying a calibration reference file on page 5 20 The Automatic Calibration Settings dialog box is displayed again see Figure 5 7 on page 5 30 7 Repeat step 5 and step 6 to add all needed masses 8 To delete masses do either of the following Select a reference then click Delete Selected Reference Click Delete All References 9 Enter Reference Matching Criteria Minimum Intensity P...

Page 237: ...for this calibration to be successful Maximum Outlier Error m z or ppm Tolerance within which all matched peaks must fall for this calibration to be successful NOTE If the software finds fewer than three matches the maximum Outlier Error is not significant Masses are labeled with the specified reference masses 12 Click Save Settings to save the automatic calibration settings reference masses match...

Page 238: ...State then select On NOTE The Auto Calibrate State command setting is stored in a DAT file That is if you turn on this command then close the DAT file the command is on the next time you open the DAT file NOTE The Auto Calibrate State command is dimmed unless you specified automatic calibration settings in the data file See Section 5 4 2 Importing and Specifying Automatic Calibration Settings 4 Di...

Page 239: ... constants calibrated mass and fit errors Successful or failed calibration message Applying auto calibration settings to other files To apply auto calibration settings to other files 1 Extract the SET information from the data file containing the auto calibration settings by selecting Settings from the File menu then selecting Save Processing Settings As For more information see Section 1 6 5 Extr...

Page 240: ...ding The centroid spectrum is displayed with a CT trace label see Figure 5 10 The height of each vertical bar corresponds to the original peak area Figure 5 10 Centroid Spectrum 4 To return to the original trace see Returning to the original spectrum on page 5 3 NOTE If the original spectrum is displayed in vertical bars instead of lines select Graphic Options from the Display menu click the trace...

Page 241: ... protein Mass Deconvolution Use when you have a spectrum with clearly resolved multiply charged peaks You specify the m z values for the peaks to include This function requires at least two adjacent peaks within the same charge envelope Convert to Zero Charge Spectrum Use when you have a spectrum with overlapping charge envelopes or a noisy baseline You specify a mass range for the peaks to includ...

Page 242: ...5 11 is displayed 4 In the Spectrum window right click drag one multiply charged peak 5 Right click drag a second multiply charged peak adjacent to the first selected peak and in the same envelope of charged peaks Mass Charge values are entered in the list box Figure 5 11 Multiply Charged Deconvolution Dialog Box ...

Page 243: ...e calculation This range is used for two purposes If you selected Automatic mode the software searches this range for other peaks that are in the same charge series as the peaks selected above The software includes these other peaks in the calculation to improve signal to noise ratio and the accuracy of the calculation Regardless of mode the software uses all data points in this range to construct...

Page 244: ... the Select Spectrum dialog box 13 Click OK Converting to zero charge To convert to a zero charge spectrum 1 Activate the Spectrum window 2 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 3 From the Process menu select Multiple Charge then select Convert to Zero Charge Spectrum The Zero Charge Spectrum Conversion dialog box Figure 5 12 on pa...

Page 245: ...t at which to perform the calculation 0 1 for resolved isotope peaks 1 0 for unresolved isotope peaks 1 0 for noisy trace Figure 5 12 Zero Charge Spectrum Conversion Dialog Box 6 Select or type the mass of the adduct ion to use in the calculation The mass of a proton H is selected by default 7 Click OK The deconvoluted trace with a DECONV trace label replaces the original trace 8 To return to the ...

Page 246: ...sian method Noise removal Procedure To noise filter or smooth the display 1 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 2 From the Process menu select Noise Filter Smooth The Noise Filter Smooth dialog box Figure 5 13 is displayed Figure 5 13 Noise Filter Smooth Dialog Box NOTE The smoothing filtering method selected in this dialog box i...

Page 247: ...ay yield better results Noise Filter NF May affect peak resolution Specify a Correlation Factor of 0 to 1 0 Settings from 0 5 to 0 7 yield acceptable results for most data A setting close to 1 0 yields a higher degree of noise reduction If applying the Noise Filter with a certain Correlation Factor does not yield the necessary noise removal return to the original trace see Returning to the origina...

Page 248: ... then removes the specified number of standard deviations of noise This method slightly affects peak intensity and removes peaks with a signal to noise ratio less than the specified standard deviation In general If you set White Noise Std Dev to removed is 1 68 2 95 3 99 If applying Noise Removal with a certain Std Dev does not yield the necessary noise removal return to the original trace see Ret...

Page 249: ... in which you want to perform the offset NOTE You can select a Chromatogram or a Spectrum window If you do not activate the correct type of window before performing the next step the software does not select values when you click drag on the trace For example you must select a Spectrum window before starting baseline offset on a spectrum 2 Select Duplicate Active Trace from the Display menu to kee...

Page 250: ...offset The selected value is displayed in the Right Baseline field 6 To limit the baseline offset to the area between the two selected points select Only Apply from L to R Baseline Midpoint To perform the baseline offset on the entire x axis deselect Only Apply from L to R Baseline Midpoint 7 Click OK The offset baseline trace is displayed with a BO trace label 8 To return to the original trace se...

Page 251: ...line that is not at 0 Some measurements for example Centroid peak detection value or the Peak Height in the Resolution calculator are derived from a peak height measured from 0 NOTE If you are analyzing Mariner data baseline correction is typically not needed If you baseline correct Mariner data note that due to the shorter flight times and fewer data points associated with Mariner data baseline c...

Page 252: ...arameters Troubleshooting Returning to the original spectrum Description The Advanced Baseline Correction feature corrects the baseline by Iteratively estimating baseline amplitude at regularly spaced intervals throughout the spectrum Smoothly connecting the calculated baseline points Removing the calculated baseline from the spectrum Figure 5 15 Advanced Baseline Correction x x x x x x x x x x x ...

Page 253: ...is function is iterative it may take several seconds to complete and typically takes longer for narrower peaks Correcting the baseline To correct the baseline 1 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 2 From the Process menu select Advanced Baseline Correction The Advanced Baseline Correction dialog box Figure 5 16 is displayed Figur...

Page 254: ...Width at half height Value the software uses to estimate the baseline amplitude at regularly spaced points in the spectrum The number of regularly spaced points used is derived using the Peak Width parameter and the Flexibility parameter The illustration below shows how using a higher number of points estimate the baseline accomplished by using higher Peak Width and Flexibility settings can affect...

Page 255: ...ue for example 10 times the width of widest peak to increase processing speed If the baseline changes sharply across the spectrum set a smaller value closer to the narrowest peak width If the baseline is broad or gently sloping set a larger value for example 10 to 20 times the width of the actual peak width To determine the number of points across a peak change the trace display from Line to Verti...

Page 256: ...o 1 0 The default value of 0 5 works best for most applications A value closer to 0 reduces flexibility and provides a smoother more generalized baseline correction see below A value closer to 1 provides more localized baseline correction by using a larger number of points to estimate the baseline see below If the baseline rises adjacent to peaks after baseline correcting decrease the Flexibility ...

Page 257: ...orrects 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 For protein and tryptic digest peptide spectra use 0 to 0 5 If the baseline after correction is too high increase the Degree value see below A lower Degree setting allows the ...

Page 258: ...ows a rising peak cluster Peak Width Average peak width Flexibility 0 5 Degree 0 5 Baseline is gently curving Peak Width 10 to 20 times peak width Flexibility 0 Degree 0 5 decrease further to raise the baseline Baseline rise should be ignored and treated as signal Peak Width Half of the width of the cluster to correct in the illustration shown if the width of the cluster is 200 set Peak Width to 1...

Page 259: ...fter the correction a hump under the peak adjust the following parameters in the order listed Decrease Flexibility Decrease Degree Decrease Peak Width NOTE Lower Peak Width values increase the time needed for processing Returning to the original spectrum To return to the original trace see Returning to the original spectrum on page 5 3 ...

Page 260: ...he Low Mass Gate spike and background in the low mass range Truncating To truncate spectra 1 Display the spectrum to truncate 2 Select Duplicate Active Trace from the Display menu to keep the original data displayed after processing 3 From the Process menu select Truncate Spectrum The Truncate Spectrum dialog box Figure 5 17 is displayed Figure 5 17 Truncate Spectrum Dialog Box 4 Type the starting...

Page 261: ...ak Figure 5 18 includes a Low Mass Gate spike Because the Low Mass Gate spike is the most intense peak in the spectrum it is identified as the base peak and All other peaks in the spectrum are scaled as a percentage of the base peak A default peak detection threshold Base Peak Intensity is set to 1 percent of the base peak One percent of an intense peak yields a high default threshold and many pea...

Page 262: ...Chapter 5 Examining Spectrum Data 5 58 Applied Biosystems 5 Figure 5 19 Truncated Spectrum Low Mass Gate Spike Eliminated ...

Page 263: ...te is present each charge state is converted appropriately and the total range of the converted spectrum is approximately 1 2 times the m z of the highest m z present Requirements Use this function only on isotopically resolved data that is labeled with the correct charge state Peaks with low signal to noise ratios may be labeled with incorrect charge states Set peak detection thresholds to disreg...

Page 264: ... Adduct text box type or select the adduct that is the charge carrying species in the spectrum you are examining 6 Select the polarity for the converted spectrum You must select the same charge as the spectrum you are evaluating CAUTION If you do not select the same charge as the spectrum you are evaluating an incorrect mass is reported 7 Click OK The converted spectrum is displayed with an SC tra...

Page 265: ...onversion Before conversion Figure 5 21 the spectrum includes 2 and 3 charged species of neurotensin 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 Figure 5 22 Spectrum After Single Charge Conversion Neurotensin multiply charged species Neurotensin singly charged species ...

Page 266: ...aturation correction is not supported for Mariner RST or DAD data Function The AutoSaturation Correction function mathematically corrects for signal saturation of the Mariner detector system to provide optimum mass accuracy The AutoSaturation Correction feature is turned on by default and automatically corrects the data when you open a Mariner data file in Data Explorer Leave this feature turned o...

Page 267: ...e Mariner Instrument Control Panel even when Saturation Correction is turned on Saturation requires information about the pulser frequency used to acquire the data and this information is not stored in RST files saved from the Instrument Control Panel Trace label When AutoSaturation Correction is turned on spectra in the data file are displayed with an ASC AutoSaturation Correction trace label ...

Page 268: ...m this function on spectra from different data files copy a spectrum trace from one data file to another See Section 2 5 2 Copying Traces from Multiple Data Files to a Window for information on copying traces 2 Activate the Spectrum window that contains the two traces of interest 3 Process the traces as needed 4 Click the first trace in the Spectrum window to make it the active trace NOTE The Dual...

Page 269: ...ic Dialog Box 6 Set the Mass Tolerance within which data points from the different traces will be considered as the same mass 7 Select Add or Subtract for Operation 8 Select Add New Trace or Replace Active Trace for the result trace 9 Click OK Absolute intensities of the two traces are added or subtracted and the result trace is displayed with ADD or SUB in the trace header ...

Page 270: ...Chapter 5 Examining Spectrum Data 5 66 Applied Biosystems 5 ...

Page 271: ...ons 6 1 Using the Elemental Composition Calculator 6 2 6 2 Using the Isotope Calculator 6 13 6 3 Using the Mass Resolution Calculator 6 20 6 4 Using the Signal to Noise Ratio Calculator 6 23 6 5 Using the Ion Fragmentation Calculator 6 25 6 6 Using the Elemental Targeting Application 6 31 6 7 Using the Macro Recorder 6 34 ...

Page 272: ...r amino acid compositions for a given mass The application then generates a theoretical isotope pattern using the Mass Resolution specified in Basic Peak Detection settings compares each observed mass and isotope pattern to the theoretical mass and isotope pattern for each possible composition and reports an isotope match score that reflects how closely they match Determining identity of fragment ...

Page 273: ...ne elemental composition 1 Display the spectrum containing the peak of interest 2 Click the Spectrum window to activate it 3 From the Applications menu select Elemental Composition The Elemental Composition Calculator dialog box Figure 6 1 is displayed Figure 6 1 Elemental Composition Calculator Dialog Box ...

Page 274: ...tal comparisons and is also multiplied by 2 and used as the window for isotope peak comparison because related isotope peaks will usually have lower mass accuracy than monoisotopic peak Setting Tolerance too low can result in a falsely low Isotope Match Score see page 6 6 6 Specify the Mass Type to calculate 7 If the displayed Mass Peak Resolution is not appropriate for this calculation change the...

Page 275: ...n State to calculate If you are calculating composition for Intact molecules Use the default of Even Only Fragment ions Set to Both Odd and Even if you are analyzing mass differences or absolute masses NOTE You may be able to refine results for fragment ions by setting Electron State to Odd Only 13 Click OK Calculating 14 Click Calculate To cancel a lengthy calculation click Cancel Calc Results Th...

Page 276: ...t mass is a protonated molecular ion M H the DBE value includes 0 5 and corresponds to an even electron state To convert the reported DBE value to the actual number of double bonds or rings add 0 5 For example H2C NH2 has an M H total mass of 30 0344 and a reported double bond equivalent of 0 5 with an actual number of double bonds of 1 Formula Elemental composition for each mass Isotope Match Sco...

Page 277: ...ow with an ISO trace label and the elemental formula To return to the original trace see Returning to the original spectrum on page 5 3 6 1 2 Setting Limits This section includes Setting limits for existing elements Adding new elements and setting limits Setting limits for other result types Setting limits for existing elements To set limits for existing elements 1 To set limits for Elemental resu...

Page 278: ...log Box NOTE Ignore the column of check boxes to the left of the Isotope column if it is displayed 3 Change the Minimum and Maximum number of occurrences for the element as needed NOTE The software ignores changes you make to the individual isotope minimum and maximum values 4 Click OK 5 Change limits for other elements as needed then click OK to return to the Elemental Composition Calculator ...

Page 279: ...tting limits To add new elements and set limits 1 To add new elements and set limits for Elemental results click Element Limits in the Elemental Composition Calculator dialog box The Limits dialog box is displayed Figure 6 5 Figure 6 5 Element Limits Dialog Box 2 Click The Periodic Table Figure 6 6 is displayed ...

Page 280: ...Chapter 6 Using Tools and Applications 6 10 Applied Biosystems 6 Figure 6 6 Periodic Table 3 Click an element to select it and to display the Isotope dialog box Figure 6 7 ...

Page 281: ...displayed 4 Change the Minimum and Maximum number of occurrences for the first isotope of the element as needed NOTE The software ignores changes you make to the individual isotope minimum and maximum values 5 Click OK two times to return to the Element Limits dialog box 6 Repeat step 2 through step 5 to add other elements as needed then click OK to return to the Elemental Composition Calculator ...

Page 282: ...the Elemental Composition Calculator dialog box see Figure 6 1 on page 6 3 The Limits dialog box for the selected Result Type is displayed Figure 6 8 shows the Amino Acid Limits dialog box Figure 6 8 Amino Acid Limits Dialog Box 2 Change the Min Number and Max Number for the compound for which you are setting limits then click OK 3 Change limits for other entries as needed then click OK to return ...

Page 283: ...ompare or overlay the theoretical distribution with your observed distribution Using the Isotope Calculator To use the Isotope calculator 1 Display the spectrum containing the observed isotope distribution 2 Click the Spectrum window to activate it 3 From the Applications menu select Isotope Calculator The Isotope Calculator dialog box Figure 6 9 is displayed Figure 6 9 Isotope Calculator Dialog B...

Page 284: ...element from the Periodic Table The first letter of two letter elemental symbols must be capitalized Spaces do not matter Amino Acid Sequence One letter or three letter amino acid codes The first letter of amino acid codes must be capitalized Spaces do not matter NOTE Do not include elemental codes when you type the sequence for example to specify water of hydration Invalid results may be generate...

Page 285: ... Group Count Charge field determines the number of groups to add or subtract and the charge state to calculate When Add Subtract is disabled the Charge field determines the charge state to calculate Whether to add or subtract the specified number of elements or groups NOTE If you specify Subtract the group to subtract must be present in the formula you specify in step 5 For more information see Ad...

Page 286: ...he Spectrum window with an ISO trace label and the elemental formula or the amino acid DNA or RNA sequence The result is displayed in the Result tab in the Output window For more information see Evaluating traces on page 6 18 Add and subtract examples The following examples illustrate how the software adds and subtracts groups to or from an elemental formula and how it applies the charge state fie...

Page 287: ...these parameters specified The software performs this calculation Formula C42H60O30Na2 C42H60O30Na2 H Group type H Group Count Charge 1 Subtract To calculate a theoretical isotope distribution for β cyclodextrin with 2 charge state m z 522 With these parameters specified The software performs this calculation Formula C42H60O30 C42H60O30 2 Group type deselected Charge 2 Add Formula entry is case se...

Page 288: ...place mode set to Replace the isotope trace replaces the active trace For more information on Replace mode see Setting the Replace mode on page 2 17 To evaluate the trace you can Overlay theoretical and observed isotope patterns See Section 2 4 8 Overlaying Traces for information Add new traces and calculate the isotope multiple times using different settings then compare or overlay the different ...

Page 289: ... The results of the calculation are displayed in the Result tab of Output window Figure 6 11 Figure 6 11 Isotope Calculation Results in Output Window If results are not calculated If results are not calculated you may have tried to remove a group that is not present in the formula ...

Page 290: ...riner data are typically at 0 Baseline correction is not necessary 3 From the Tools menu select 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 w...

Page 291: ... resolution by doing one of the following Type in a Mass Charge value In the Spectrum window right click drag over the peak for which you want to calculate resolution The mass is automatically entered in the Resolution Calculator dialog box NOTE If you right click drag over more than one peak the mass of the highest peak is used 6 Click OK FWHM 50 peak height ...

Page 292: ...Chapter 6 Using Tools and Applications 6 22 Applied Biosystems 6 The result is displayed in the Output window Figure 6 13 Figure 6 13 Resolution Calculator Results Resolution results calculator ...

Page 293: ...ak for calculation and the software automatically calculates the average noise across the spectrum Manual You specify the peak and the baseline region for calculation For accurate results this method requires a flat non rising baseline that does not include peaks The manual method is useful when evaluating a narrow region around a peak to determine the relative significance of a peak Calculating s...

Page 294: ...the average noise across the entire spectrum The noise calculation is not affected by the presence of peaks or a poor baseline Manual Right click drag across the apex of the peak for signal to noise calculation Right click drag across a flat region of the baseline to use to determine the RMS noise for the calculation NOTE If you enter a baseline value instead of right click dragging the software u...

Page 295: ...ner data PSD fragments you may see in Voyager data 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 Fragmentation calculator To use the Ion Fragmentation Calculator 1 Display the spectrum of interest 2 Click the Spectrum window to activate it 3 From...

Page 296: ... 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 5 Select the N terminus and C terminus options for the sequence 6 Specify the cysteine modification and specify the mass type average or monoisotopic for the calculated fragment ion masses ...

Page 297: ...lts for internal fragments 9 If you are analyzing Mariner data select Calculate multiple charge states if desired then specify the maximum charge state to calculate up to 12 10 Type the Mass Tolerance to use when labeling peaks Masses that fall outside the tolerance are not labeled 11 To add the labels generated by this operation to the list of available user labels select Append new labels to exi...

Page 298: ...isplayed Figure 6 17 User Defined Amino Acids Dialog Box 14 Add amino acid definitions and codes as needed NOTE You cannot modify pre defined amino acids User defined amino acids are not saved when you close the Data Explorer software 15 Click Close Calculating the fragment ions 16 Click Induce Fragmentation Results are displayed in the Ion Fragmentation Calculator dialog box Figure 6 18 ...

Page 299: ...PPPPPPAR Results Results are displayed in the Ions table Lists the masses for each fragment and ion type Internal fragments table Lists possible internal sequences if you enabled this option in the Options dialog box see Figure 6 16 on page 6 27 Click Clear Table Info to clear results You can change options and recalculate ion fragmentation results ...

Page 300: ...t The Label Peaks function creates User Labels in the data file To view select Peak Label from the Peaks menu then select User Label Setup Click Save As to save the labels in a LBS file for use with other data files For more information see Section 3 5 3 Setting Custom Peak Labels To display the original labels select Peak Label from the Peaks menu then deselect User Labels Creating a calibration ...

Page 301: ...to screen a spectrum for the presence of specific chemical compounds Using the Elemental Targeting application To use the Elemental Targeting application 1 Display the spectrum of interest 2 Click the Spectrum window to activate it 3 From the Applications menu select Elemental Targeting The Elemental Targeting dialog box Figure 6 20 is displayed 4 Enter the Mass Tolerance for the evaluation 5 If t...

Page 302: ...matter for formula The first letter of two letter elemental symbols must be capitalized for example Na To ensure a better match between theoretical and observed isotopes include the appropriate number of protons in the formula you enter for multiply charged ions Figure 6 20 Elemental Targeting Dialog Box 7 Click Calculate ...

Page 303: ...tion you specified Charge Charge of the observed peak Isotope Match Score Number between 0 0 and 1 0 that reflects how well the observed peak matches the theoretical formula based on mass and isotope pattern A higher number represents a better match Isotope Match Intensity Peak area counts that overlaps between the observed isotope pattern and the theoretical isotope pattern A higher number repres...

Page 304: ...on includes Before using the macro recorder Recording a macro Assigning macros to buttons Running a macro Deleting a macro Advanced macro editing Importing or Exporting Macros in DATAEXPLORER VB6 Running macros automatically when opening and closing files 6 7 1 Before Using the Macro Recorder Importing macros provided Macros are provided with the Data Explorer software but are not available for us...

Page 305: ... been assigned to it For a description of a toolbar button place the cursor on the toolbar button The name you assign to the macro or the default macro name is displayed below the button Functions not supported Most commands in the Data Explorer software can be included in a macro that you create with the Macro Recorder The following menu commands are not supported by the Macro Recorder Menu Comma...

Page 306: ...tion Mass Calibration Revert to instrument calibration Mass Calibration Edit Create Reference File Dual Spectral Trace Arithmetic Multiple Charge Mass Deconvolution Peaks Peak Detection Peak Label Insert Peak chromatogram Tools Processing History Options Customize Toolbar Customize ToolMenu Macro commands including Automatic Macros Applications Elemental Targeting Ion Fragmentation Calculator Wind...

Page 307: ...ialog Box 3 Type a name and a description if desired 4 Click OK 5 Select the commands you want to automate with the macro For example select Noise Filter Smooth from the Process menu select the smoothing method specify the associated parameter then click OK Not all functions in Data Explorer are supported See Functions not supported on page 6 35 6 From the Tools menu select Stop Macro Recording Th...

Page 308: ... Explorer project For information see Section 6 7 7 Importing or Exporting Macros in DATAEXPLORER VB6 Assigning a macro to a button You can assign macros to 10 buttons To assign a macro 1 From the Tools menu select Assign Macro The Assign Macro dialog box Figure 6 24 is displayed Figure 6 24 Assign Macro Dialog Box 2 Select the button to assign from the list on the left and the macro to assign fro...

Page 309: ...mmand Using a toolbar button To run a macro using the toolbar button 1 Open the data file on which you want to run the macro 2 Click the button assigned to the macro you want to run Hint Place the cursor on a macro button to display the name of the macro assigned to the button The macro executes Using the menu command To run a macro using the menu 1 Open the data file on which you want to run the ...

Page 310: ...ro contains a syntax error it may cause the Data Explorer software to close unexpectedly If this occurs restart the Data Explorer software and examine the macro in the Visual Basic Editor See Section 6 7 6 Advanced Macro Editing Macro lines containing syntax errors are displayed in color in the Visual Basic Editor For information on correcting errors refer to the online help available in the Visua...

Page 311: ...macro 1 From the Tools menu select Macros The Macros dialog box Figure 6 26 is displayed Figure 6 26 Macros Dialog Box 2 Select the macro to delete from the list 3 Click Delete NOTE Other buttons on this dialog box are for advanced editing Refer to the online help available within the Visual Basic Editor ...

Page 312: ...g the Macro Recorder in Data Explorer are stored in Module folders in the Project Explorer window Displaying scriptable objects To display a list of the scriptable objects available within Data Explorer select Object Browser from the View menu in the Visual Basic Project Explorer window For information on using the Visual Basic Editor refer to the online help in the Visual Basic Editor Numbering s...

Page 313: ...n stand alone ASCII format BAS and FRM files in C MARINER PROGRAM MACROS or C VOYAGER MACROS and are not incorporated into the DATAEXPLORER VB6 file automatically To make the new macros available for use import them into the DATAEXPLORER VB6 file Importing NOTE You can also import by opening NT Explorer then click dragging the files into the DataExplorerProject displayed in the Visual Basic Editor...

Page 314: ...ms folder in the DataExplorerProject All macros imported into the DataExplorerProject are displayed in the list of macros you can assign in the Data Explorer software See Section 6 7 3 Assigning Macros to Buttons Exporting To export macros 1 Access the VBA Editor from Data Explorer as described above 2 From the File menu select Export File 3 Select the BAS or FRM file to export then click Save NOT...

Page 315: ...ated when you open or close a data file For information on creating macros see Section 6 7 2 Recording a Macro To set up automatic macros 1 From the Tools menu select Automatic Macros The Automatic Macro Setup dialog box Figure 6 26 is displayed Figure 6 27 Automatic Macro Setup Dialog Box 2 Select File Open Macro or File Close Macro as desired then select the macro to run when you open or close a...

Page 316: ...Chapter 6 Using Tools and Applications 6 46 Applied Biosystems 6 ...

Page 317: ...roving 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 7 2 Voyager Data Examples 7 11 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 Detecting Peaks from Complex Digests 7 18 ...

Page 318: ...m By creating an extracted ion chromatogram for a specific mass you can screen out ion contribution from masses that you are not interested in Creating an extracted ion chromatogram The following example illustrates how you can create an extracted ion chromatogram to improve the signal to noise ratio for reserpine 609 Da 1 Display the data file 2 In the Chromatogram window click in the toolbar or ...

Page 319: ...he improved signal to noise ratio in the extracted ion chromatogram for three replicate loop injections Figure 7 1 Improving Signal To Noise Ratio with an Extracted Ion Chromatogram Original TIC containing all masses Extracted ion with improved signal to noise ratio chromatogram for 609 Da ...

Page 320: ...and comparing the extracted ion chromatograms to the original TIC containing the unresolved peaks You can then create a combined spectrum from each extracted ion chromatogram to evaluate the spectral data Creating a combined spectrum The following example illustrates how to deconvolute two unresolved chromatographic peaks in a tryptic digest of cytochrome c 1 Display the data file 2 In the Chromat...

Page 321: ... Spectrum for Unresolved Peaks in Cytochrome C Creating extracted ion chromatograms To create extracted ion chromatograms 1 With the Chromatogram window activated click in the toolbar two times to add two traces Click an added trace to activate it 2 In the Spectrum window right click drag over the first peak The extracted ion chromatogram is displayed 3 In the Chromatogram window click the second ...

Page 322: ...vate the Spectrum window then click in the toolbar two times to add two traces 2 Right click drag over the first half of the mass range in the 410 extracted ion chromatogram 3 Activate the second trace in the Spectrum window 4 Right click drag over the second half of the mass range in the 723 extracted ion chromatogram Extracted ion chromatograms with deconvoluted peaks Original TIC containing unr...

Page 323: ...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 Figure 7 5 Combined Spectra for Deconvoluted Peaks Both combined spectra contain a peak at 391 Da ...

Page 324: ...ed at 391 Da Figure 7 5 on page 7 7 is eliminated To subtract spectra 1 Activate the first extracted ion chromatogram in Chromatogram window see Figure 7 4 on page 7 6 2 From the Process menu select Add Subtract Spectra 3 Click then right click drag over the peak in the extracted ion chromatogram The spectrum range is displayed in the Spectra To Be Added list in the Add and Subtract Spectra dialog...

Page 325: ... User s Guide 7 9 7 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: ...below and evaluate the signal Creating extracted ion chromatogram To create the extracted ion chromatogram right click drag over the peak at 391 Da in the spectrum trace NOTE You can also create an extracted ion chromatogram by selecting Trace from the Display menu selecting Extracted Ion then entering a mass Figure 7 8 illustrates the extracted ion chromatogram for mass 391 The signal is relative...

Page 327: ...th partially resolved peaks that represent two compounds If Adjust the following Peaks represent two compounds and you want both peaks labeled In the Peak Label dialog box select Allow Overlapping Peak Labels OR On the Basic Settings tab in Peak Detection set Max Peak Area to 0 then adjust the Base Peak Intensity until peaks are detected OR On the Peak Processing tab in Peak Detection change the d...

Page 328: ...unds Minor Component Not Detected Adjusting peak detection To adjust peak detection 1 Click in the toolbar or select Peak Detection from the Peaks menu The Spectrum Peak Detection Setup dialog box is displayed with the Basic Settings tab Figure 7 10 displayed Figure 7 10 Spectrum Peak Detection Setup Basic Settings Tab ...

Page 329: ...om 1 the default to 0 and the Base Peak Intensity from 0 the default to 1 Click the Peak Processing tab and change the default Integration Baseline Setting from Valley to Valley to Valley to Baseline 3 Click OK Figure 7 11 shows the partially resolved peaks that are now labeled Figure 7 11 Partially Resolved Peaks Labeled ...

Page 330: ...librating without baseline correcting and deisotoping For optimum mass accuracy baseline correct and deisotope a spectrum before calibrating Figure 7 12 shows a spectrum before calibration Figure 7 12 Spectrum Before Calibration Figure 7 13 shows a spectrum that has been calibrated without initial baseline correction and deisotoping Mass accuracy is not acceptable Figure 7 13 Calibration Without B...

Page 331: ...on see Section 5 8 2 Using Baseline Correction Deisotoping 3 From the Peaks menu select Peak Deisotoping The Deisotoping dialog box Figure 7 14 is displayed Figure 7 14 Deisotoping Dialog Box 4 For this example spectrum specify H for Adduct and C6H5NO for Generic Formula 5 Click OK Figure 7 15 shows the baseline corrected deisotoped spectrum before calibration For more information on deisotoping s...

Page 332: ...use for calibration Click OK 2 From the Process menu select Mass Calibration and then select Manual Calibration The Manual Calibration dialog box is displayed Figure 7 16 Figure 7 16 Manual Calibration Dialog Box 3 Click then select the VOYAGER REF calibration reference file 4 Enter Reference Matching and Calibration Criteria NOTE For descriptions of calibration parameters see Section 5 3 Manual C...

Page 333: ... peaks will match You can also add peaks to the Peak Matched list by right click dragging on a peak in the spectrum then selecting the mass from the reference Peak Information dialog displayed Click Solve and Plot after manually adding masses The spectrum is calibrated and displayed with an MC trace label The calibration statistics are displayed in the Output window Applying 6 If the calibration s...

Page 334: ...ic peaks are detected for proper deisotoping Deisotope to identify isotope peak clusters The deisotope function amplifies peak intensity based on the total area of all peak areas in the isotope cluster It does not amplify noise peaks Increase peak detection thresholds to eliminate noise peaks Figure 7 18 represents a spectrum trace from a digest Overlapping Peak Labels are enabled to illustrate th...

Page 335: ...Smooth dialog box Figure 7 19 is displayed Figure 7 19 Noise Filter Smooth Dialog Box 3 Select Default Smoothing or Noise Filter with a Correlation Factor of 0 7 then click OK For more information see Section 5 7 Noise Filtering Smoothing Setting detection thresholds 4 Click in the toolbar or select Peak Detection from the Peaks menu The Spectrum Peak Detection Setup dialog box is displayed with t...

Page 336: ...Chapter 7 Data Explorer Examples 7 20 Applied Biosystems 7 Figure 7 20 Spectrum Peak Detection Setup Basic Settings Tab 5 Click Use Advanced Settings The Advanced Settings tab is displayed Figure 7 21 ...

Page 337: ...ea to 0 7 Click the Basic Settings tab see Figure 7 20 on page 7 20 then set Max Peak Area to 0 or 0 1 8 Click Apply Note that many peaks are added to the peak list in the Output window For more information see Section 3 2 Peak Detection Deisotoping 9 From the Peaks menu select Peak Deisotoping The Deisotoping dialog box Figure 7 14 is displayed ...

Page 338: ... detection thresholds 12 In the Basic Settings tab in Global Thresholds see Figure 7 20 on page 7 20 increase Max Peak Area to a setting between 0 1 and 1 until the peaks of interest are detected and noise is screened out Figure 7 23 shows the trace after deisotoping Note that many fewer peaks are detected By further fine tuning Max Peak Area you can screen out additional noise peaks Figure 7 23 D...

Page 339: ...ons 8 1 Displaying PSD Data 8 2 8 2 Applying Fragment Labels 8 8 8 3 Calibrating a PSD Spectrum 8 10 8 3 1 Checking Peak Detection 8 11 8 3 2 Calibrating 8 12 8 3 3 Creating PSD CAL Files and Applying to Other Data Files 8 20 8 3 4 Creating PSD Calibration Reference REF Files 8 21 8 3 5 Changing the Precursor Mass 8 23 ...

Page 340: ...xplorer software until you stop the PSD experiment in the Instrument Control Panel Error messages are displayed if you try to open the file For more information see the Voyager Biospectrometry Workstation User s Guide To display PSD data 1 Open the PSD data file of interest in the Data Explorer software as described in Section 2 1 Opening and Closing Data Files NOTE PSD data files are named with a...

Page 341: ...ces click and Segments are displayed in the order in which they were acquired NOTE If these buttons are not displayed in the toolbar you can add them See Section 1 4 3 Customizing Toolbars Displaying multiple segment traces To display multiple segment traces 1 Click in the toolbar three times to add three Not Used traces 2 From the Process menu select PSD Processing ...

Page 342: ... 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 This value reflects the maximum mass of the segment that will be included in the composite spectrum The mass range included in the segment is approximately 15 percent higher than the Max Stitch Mass Optimum focus and resolution is achieved for fragment ions c...

Page 343: ...der of acquisition For example if you listed Segments 1 through 5 for acquisition but acquired Segments 1 3 and 5 you would see three entries above that correspond to the three acquired segments 3 In the Spectrum window click the first Not Used trace 4 In the PSD Processing dialog box double click the Entry number of the segment to add 5 Repeat step 3 and step 4 to display additional segments 6 To...

Page 344: ... determines the region of each segment to include in the composite spectrum as illustrated in Figure 8 3 on page 8 7 Applies baseline correction and two standard deviation noise removal to the composite region of each segment Stitches the composite regions together and displays the composite spectrum PSD calibration equation The equation that the Voyager software uses to calculate the mass for fra...

Page 345: ...ch the segment was acquired and the mass of the precursor ion mp Figure 8 3 Figure 8 3 Portions of Segment Traces Included in the Composite Spectrum PSD Internal Standard Calibration Constant Value Used One point α Calculated from standard mass β and γ 0 Two point or three point α and β Calculated from standard masses γ 0 More than three point α β γ Calculated from standard masses Seg 1 Seg 2 Seg ...

Page 346: ...For detailed information on using the Ion Fragmentation calculator see Section 6 5 Using the Ion Fragmentation Calculator Applying labels To apply fragment labels to PSD spectra 1 From the Applications menu select Ion Fragmentation Calculator The Ion Fragmentation Calculator dialog box Figure 8 4 is displayed Figure 8 4 Ion Fragmentation Calculator Dialog Box ...

Page 347: ...5 Click Label Peaks The ion peaks specified in Options are labeled on the trace if they are present Hint To more selectively apply labels decrease the Mass Tolerance in the Options dialog box Hint This function creates User Labels in the data file To view select Peak Label from the Peaks menu then click User Label Setup Click Save As to save the labels in a LBS file for use with other data files F...

Page 348: ...u use to acquire unknown PSD samples Internally calibrate an unknown PSD data file by specifying known monoamino acid fragment ions as the peaks to match during calibration Overview of creating a PSD CAL file To create a PSD CAL file Acquire a peptide standard with a known sequence in PSD mode Check peak detection as described in Section 8 3 1 Checking Peak Detection Create a calibration reference...

Page 349: ...r different mass ranges You can fine tune ranges and Filter Width settings in ranges to optimize detection For more information see Detection Ranges on page 3 28 Fragment ion peaks in segments collected with lower PSD Mirror Ratios are broader and include more data points Use a higher Filter Width setting for these segments Within a segment resolution increases with increasing mass as the flight t...

Page 350: ... result spectrum or a composite spectrum you have accessed from the Processing History command on the Display menu For more information see Section 2 4 7 Recalling and Rearranging Traces Processing History 2 From the Peaks menu select Peak Label then select the Mass Label Type peak apex or peak centroid to use for calibration Click OK 3 From the Process menu select Mass Calibration then select PSD...

Page 351: ...ion reference REF file that specifies the peak type for reference masses as Resolved Isotope Mass even if they are not resolved isotopes The calibration routine checks peak width to determine if a peak matches a Resolved Isotope Mass or an Average mass If narrow peaks are specified as Average Masses in the calibration reference file the software mistakes these narrow peaks as isotopically resolved...

Page 352: ...ntensity or Relative Area Mass Tolerance Peaks must be within this tolerance of the theoretical mass to be considered a match Select the unit m z or ppm 6 Select the Peak Weighting Factor If the calibration includes more than two points you can apply the following weighting factors to fit points to the curve None All peaks weighted equally Inverse Peak Width Narrower peaks are weighted more than b...

Page 353: ... mass range in all segments in the DAT file NOTE The entire mass range of each spectrum in the DAT file is checked not just the mass range included in the composite spectrum and even if the spectrum is not currently displayed in the Spectrum window For comparison the difference between the reference mass in the calibration reference file and the observed peak mass is displayed Hint You can sort th...

Page 354: ... on page 8 19 To manually select the reference mass for a peak 1 Right click drag over the peak of interest The Reference Mass dialog box Figure 8 6 is displayed and lists all masses in the calibration reference file The entry highlighted is the nearest match in the calibration reference file to the selected peak that is within the mass tolerance specified Figure 8 6 Reference Mass Dialog Box ...

Page 355: ...ormation to the calibration reference file then click OK to accept the reference mass for matching The PSD Calibration dialog box is displayed again see Figure 8 5 on page 8 13 with the observed mass and the reference mass you selected displayed in the Matched Peak list 3 Repeat step 1 through step 2 until the desired peaks are in the matched list For optimum mass accuracy take at least one mass f...

Page 356: ...st Use the scroll bar to view newer statistics at the bottom of the list The spectrum is calibrated and displayed with an MC trace label The calibration statistics are displayed in the Output window Applying new constants to the data file To apply the calibration constants to each spectrum all segments in the data file click Apply Calibration All spectra in the data file are calibrated and display...

Page 357: ... add the same mass to the list multiple times Hint A calibration reference file called Immonium_Ions REF is provided with the software To optimize mass accuracy across the entire PSD composite spectrum multi point calibration Select reference peaks from a wide range of segments to ensure that high and low Mirror Ratios are represented Include peaks with masses that are substantially below the Max ...

Page 358: ... the file Hint Include a _PSD suffix when you export a PSD CAL file to help you distinguish them from non PSD CAL files For example type Cal_PSD as the file name The complete file name will be Cal_PSD CAL Applying new constants to additional files You can apply calibration constants from a CAL file to any data file To apply the new constants from a mass calibration file to a different file 1 Displ...

Page 359: ...le For detailed information on using the Ion Fragmentation calculator see Section 6 5 Using the Ion Fragmentation Calculator Using the Ion Fragmentation calculator to create a calibration reference REF file To create a PSD calibration reference file using the Ion Fragmentation calculator 1 From the Applications menu select Ion Fragmentation Calculator The Ion Fragmentation Calculator dialog box is...

Page 360: ...ically resolved and ignores the reference mass For more information on creating calibration reference files see Section 5 3 3 Creating or Modifying a Calibration Reference File REF 4 Click Induce Fragmentation Results are listed in the ions table and fragment ions are labeled if they are present in the spectrum 5 Click Create Reference File A Save As dialog box is displayed 6 Name and save the cal...

Page 361: ...agment ions improves with a different precursor mass specified Before changing the precursor mass note the following points The Change Mass function changes the value for mp in the PSD calibration equation described on page 8 6 It does not change the value for tp Use the Change Mass function only if the precursor mass used to acquire the data does not correspond to the sequence you are observing i...

Page 362: ...ration 3 Click OK 4 Click Plot to display the composite spectrum for the new mass The following occurs A new composite spectrum is generated as described in How the composite spectrum is generated on page 8 6 and displayed The PSD calibration for the data file is updated with the new precursor ion mass The new composite spectrum is displayed NOTE The Max Stitch Masses displayed in the PSD segment ...

Page 363: ...s chapter contains the following sections 9 1 Overview 9 2 9 2 General Troubleshooting 9 3 9 3 Processing Tools and Applications Troubleshooting 9 6 9 4 Calibration Troubleshooting 9 10 9 5 Printing Troubleshooting 9 14 9 6 Peak Detection and Labeling Troubleshooting 9 15 ...

Page 364: ...ng Peak detection and labeling troubleshooting Troubleshooting information is organized according to likelihood of possible cause from most likely to least likely possible cause If you are unable to solve your problem using the information in the following tables call Applied Biosystems Technical Support To reach Applied Biosystems Technical Support refer to the list of offices on the back cover o...

Page 365: ...software windows are displayed on top of the Data Explorer window or toolbar buttons or status indicators are not displayed Multiple application windows overlap Close applications not in use then minimize and maximize the Data Explorer window to refresh the display Failed to create empty document message displayed when you open a data file Chromatogram and Spectrum windows are too small Resize the...

Page 366: ...d on current trace Annotation stays in the view until you delete it even if you advance to the next spectrum Delete the annotation See Section 2 4 9 Annotating Traces Table 9 1 General Troubleshooting Mariner and Voyager Continued Symptom Possible Cause Action Table 9 2 General Troubleshooting Mariner Only Symptom Possible Cause Action Failed to open chromatogram data message displayed when you op...

Page 367: ...onversion of SPC to DAT failed A DAT file with the same file name you are specifying for the converted SPC file is open 1 Close the open DAT file 2 Use a different name for the converted SPC file to prevent the new conversion from overwriting the existing file Table 9 2 General Troubleshooting Mariner Only Continued Symptom Possible Cause Action Table 9 3 General Troubleshooting Voyager Only Sympt...

Page 368: ...ve tried to remove a group that is not present in the formula Can only remove a group that is present in the formula For information see Section 6 2 Using the Isotope Calculator All traces in an overlaid trace are not processed Only the active trace in an overlaid trace is processed Display individual traces select Use same settings for all graphs from Graphic Options then process For more informa...

Page 369: ...ing Traces Section 2 6 Saving Opening and Deleting DAT Results Section 2 8 Saving Opening and Deleting SPC Results Files Mariner Data Only Link View command does not have an effect on windows or open data files You did not select Link View for each window or data file You must select Link View for each window and each data file you want to link See Linking views on page 2 13 Table 9 4 Processing T...

Page 370: ...are labeled with an incorrect charge state 1 Set peak detection thresholds to disregard these peaks See Section 3 2 5 Charge State Determination and Examples 2 Convert the spectrum again See Section 5 10 Converting to a Singly Charged Spectrum Mariner Data Only Table 9 4 Processing Tools and Applications Troubleshooting Mariner and Voyager Continued Symptom Possible Cause Action ...

Page 371: ...tiple Charge Mass Deconvolution commands dimmed on Process menu Your system does not include the optional mass deconvolution software Contact Applied Biosystems to purchase the option Centroiding Mass Calibration Multiple Charge commands not displayed on Process menu Spectrum window not active Activate Spectrum window then select Process menu Resolution command not displayed on Tools menu Spectrum...

Page 372: ...e calibration reference file The calibration routine checks peak width to determine if a peak matches a Resolved Isotope Mass or an Average mass If narrow peaks are specified as Average Masses in the calibration reference file the software mistakes these narrow peaks as isotopically resolved and ignores the reference mass When specifying highly charged non isotopically resolved species with peaks ...

Page 373: ...red during calibration Do not include multiple entries with the same m z value in the Calibration Reference file See Section 5 3 3 Creating or Modifying a Calibration Reference File REF REF file created with Windows Notepad not listed when you select Reference file Reference file does not include REF extension Some applications automatically append a TXT extension to file names To name the file wi...

Page 374: ...ration command is dimmed when calibrating MS Method data For MS Method data calibration is valid for an individual spectrum You cannot apply the calibration from one spectrum to the entire data file No action Normal occurrence Error displayed when you import a calibration CAL file corrupted Create new CAL file See Exporting BIC MSM and CAL files on page 1 36 Importing a CAL file generated from a V...

Page 375: ... CAL file See Exporting BIC MSM and CAL files on page 1 36 Importing a CAL file generated from a Mariner data file Import a Voyager CAL Importing a CAL file generated from a data file collected in a different instrument mode Linear Reflector or PSD Import a CAL generated from a data file collected in the same instrument mode Importing a CAL file generated on a different instrument Import a CAL gen...

Page 376: ...ta Explorer Landscape option set using Printer Setup in Data Explorer Set landscape printing using Printer Settings in Windows Control Panel See Dedicating a printer to landscape orientation on page 2 35 Data file names do not print for multiple data files Some printers may not print the data file name if you select Print All Views from the File menu with more than two data files open We have obse...

Page 377: ...use Action Peaks are not detected or labeled Peaks are very close together or label is too long Zoom in on region of interest Select Allow overlapping peak labels in the Peak Label dialog box See Section 3 5 2 Setting Chromatogram and Spectrum Peak Labels Peak detection parameters in particular Filter Width not set to detect peaks Adjust parameters See Section 3 2 Peak Detection Peak filtering is ...

Page 378: ... to 0 Adjust Max Peak Area to optimize peak detection for Voyager data See Section 3 2 2 Strategy for Voyager Peak Detection 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 The Chromatogram window was active when you selected Peak Label from the Display menu Make sure the Spectrum window is act...

Page 379: ... Labels Peak label placed on peak shoulder instead of peak apex Filter Width Increment set higher than 1 Set Increment to 1 See Increment on page 3 31 Table 9 10 Peak Detection and Labeling Troubleshooting Mariner and Voyager Continued Symptom Possible Cause Action Table 9 11 Peak Detection and Labeling Troubleshooting Voyager Only Symptom Possible Cause Action Noise detected as peaks Max Peak Are...

Page 380: ...2 2 Strategy for Voyager Peak Detection Table 9 11 Peak Detection and Labeling Troubleshooting Voyager Only Symptom Possible Cause Action Table 9 12 Charge State and Isotope Determination Troubleshooting Mariner Only Symptom Possible Cause Action Known isotope labeled with incorrect charge state too low Max Charge State parameter set too low See example in Max Charge State set too low on page 3 33...

Page 381: ...nly on page 3 26 Peak List Filtering is enabled with Charge State filter enabled and set too low Disable Peak List Filtering See Filtering the spectrum peak list on page 3 42 Minimum Intensity set too high to detect other isotope peaks See example in Effect of Minimum Intensity on page 3 36 Set Minimum Intensity correctly See Advanced Settings spectrum data only on page 3 28 Filter width set too h...

Page 382: ... data only on page 3 26 Peaks are more than 1 Da apart No action Normal occurrence Peaks are not from the same isotope species No action Normal occurrence Filter width is set too high to detect other isotope peaks Adjust Filter Width See Advanced Settings spectrum data only on page 3 28 Maximum Charge State for charge state determination is set lower than the charge state of the peak Adjust the Ma...

Page 383: ... isotope peaks is detected Increase Base Peak Intensity or Max Peak Area to eliminate noise See Basic Settings spectrum data only on page 3 22 Apply noise filter See Section 5 7 Noise Filtering Smoothing Monoisotopic peak not labeled correctly Peak detection charge state parameters are not set correctly the software is identifying the tallest peak in a cluster as the monoisotopic peak Adjust param...

Page 384: ...Chapter 9 Troubleshooting 9 22 Applied Biosystems 9 ...

Page 385: ...Applied Biosystems also reserves the right to require that computer hardware and software be restored to the standard configuration prior to providing service or technical support Limited Product Warranty Limited warranty Applied Biosystems warrants that for a period of ninety 90 days from the date of installation the Data Explorer software designated for use with Mariner API TOF Workstations or V...

Page 386: ...d to the buyer Warranty claims Warranty claims must be made within the applicable warranty period or for chemicals or other consumable products within thirty 30 days after receipt by the buyer Warranty exceptions The above warranties do not apply to defects resulting from misuse neglect or accident including without limitation operation outside of the environmental or use specifications or not in ...

Page 387: ...Y OF SUCH DAMAGES INCLUDING WITHOUT LIMITATION DAMAGES ARISING FROM OR RELATED TO LOSS OF USE LOSS OF DATA FAILURE OR INTERRUPTION IN THE OPERATION OF ANY EQUIPMENT OR SOFTWARE DELAY IN REPAIR OR REPLACEMENT OR FOR LOSS OF REVENUE OR PROFITS LOSS OF GOOD WILL LOSS OF BUSINESS OR OTHER FINANCIAL LOSS OR PERSONAL INJURY OR PROPERTY DAMAGE NO AGENT EMPLOYEE OR REPRESENTATIVE OF APPLIED BIOSYSTEMS HAS...

Page 388: ...Appendix A Warranty A 4 Applied Biosystems A ...

Page 389: ...endix Data Explorer Software User s Guide B 1 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 ...

Page 390: ... low abundance such as 14 C do not affect the appearance of a mass spectrum However isotopes that occur in greater abundance such as 13 C which occurs in a natural abundance of approximately 1 1 percent1 in carbon do affect the appearance of a mass spectrum The mass spectrum of methane Figure B 1 illustrates the impact of an isotope on the appearance of a mass spectrum Methane includes a peak repr...

Page 391: ...bon atoms Therefore M 2 ions become more visible In a compound with ten carbon atoms such as decahydro naphthalene C10H18 Figure B 2 relative heights of M M 1 and M 2 peaks are 100 11 0 5 Figure B 2 Mass Spectrum of Decahydro Naphthalene Isotopic pattern in mass spectra All compounds containing carbon include molecular ions and isotopic ions that are 1 and 2 mass units higher than the molecular io...

Page 392: ...The mass range you analyze and the resolving power of the analyzer determine if you observe resolved isotope peaks in a mass spectrum Figure B 3 represents angiotensin I at a resolution of 3 000 Isotopes are fully resolved and sharp peaks are observed in the mass spectrum At lower resolutions the shape of the individual peaks becomes less pronounced or may not be differentiated at all Figure B 4 r...

Page 393: ...topes 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 Figure B 5 Unresolved Isotopes M M 1 M 2 isotopic envelope ...

Page 394: ... is used for mass labeling and corresponds to the lowest mass peak in the cluster Figure B 6 Monoisotopic Mass When isotopes are not resolved Figure B 7 the average mass is used for mass labeling and corresponds to the centroid of the unresolved peak cluster weighted average of all isotope peaks in the cluster Monoisotopic mass corresponds to lowest mass peak ...

Page 395: ...Monoisotopic and Average Masses Data Explorer Software User s Guide B 7 B Figure B 7 Average Mass Average mass corresponds to centroid of unresolved peak cluster ...

Page 396: ...9737 100 2H 2 0141 0 015 32S 31 9720 95 02 12 C 12 98 90 33 S 32 9714 0 75 13C 13 0033 1 10 34S 33 9678 4 21 14 N 14 0030 99 63 36 S 35 9670 0 02 15N 15 0001 0 37 35Cl 34 9688 75 77 16 O 15 9949 99 76 37 Cl 36 9659 24 23 17O 16 9991 0 04 39K 38 9637 93 2581 18 O 17 9991 0 200 40 K 39 9639 0 012 19F 18 9984 100 41K 40 9618 6 7302 23 Na 22 9897 100 79 Br 78 9183 50 69 28Si 27 9769 92 23 81Br 80 9162...

Page 397: ...ros This appendix includes C 1 Overview C 2 C 2 Preparing Data Before Accessing Macros C 3 C 3 Accessing the Macros C 4 C 4 Using the Ladder Sequencing Toolbox C 5 C 5 Using the Peptide Fragmentation Toolbox C 9 C 6 Using the Polymer Analysis Toolbox C 15 C 7 Using MS Fit MS Tag Toolbox C 18 ...

Page 398: ...peptide or peptide fragment spectra to perform a protein database search Importing macros provided If the macros listed above are not listed when you try to run them described in Section C 3 Accessing the Macros you must import them into the Data Explorer project For information see Section 6 7 7 Importing or Exporting Macros in DATAEXPLORER VB6 Modifying the macros The Visual Basic macros describ...

Page 399: ...elect VisualBasicEditor from the Tools menu in the Data Explorer window 2 Select References from the Tools menu in the Visual Basic Editor C 2 Preparing Data Before Accessing Macros Before you access the toolbox open data files of interest and Smooth the data if necessary to reduce noise Set optimum peak detection and labeling Set the display range or zoom as needed Resize and organize the display...

Page 400: ... macros 1 Open the Data Explorer software 2 Open a data file 3 Prepare the data as described in the previous section 4 From the Tools menu select Macros 5 In the Macros dialog box select modToolBoxPalette Toolbox_Palette then click Run The Toolbox Palette dialog box is displayed Figure 3 8 Toolbox Palette Dialog Box ...

Page 401: ...idual macro in the Macros dialog box C 4 Using the Ladder Sequencing Toolbox Use the Ladder Sequencing toolbox when you perform sequencing to label peaks with the appropriate amino acid DNA or RNA residue Labels are determined by calculating the mass differences between the peaks and comparing the values to an internal listing of mass differences and corresponding residues Running the macro To run...

Page 402: ...Spec Peak List 4 Remove peaks you do not want included in the calculation by clicking the peak in the list then clicking Delete Selected Peaks Adducts 5 Select the type of spectrum you are examining Peptide DNA or RNA 6 Under Adducts to Remove select the items you do not want included in the interpretation Figure 3 9 Ladder Sequencing Toolbox ...

Page 403: ...Mass of the current peak preceded by an equal sign 8 Click Label Peaks If Peptide is selected the software Examines the spectrum in 55 Da increments and selects the most intense ion in the range The 55 Da increment is used because it is less than the smallest mass difference related to a residue Labels mass differences plus or minus the specified Tolerance that correspond to amino acids Labels the...

Page 404: ...est mass difference related to a DNA or RNA base Labels mass differences plus or minus the specified Tolerance that correspond to DNA ACGT or RNA ACGU bases Labels the reference peak from which the mass difference was derived with an asterisk Applies the additional labels you selected under Annotate Spectrum Displaying the original labels To display the original labels select Peak Label from the P...

Page 405: ... source CID spectra to label immonium ions identify fragment ion pairs view sequences based on different reference peaks and determine if a selected peak is of a specific fragment ion category Setup To enter Setup parameters for the Peptide Fragmentation Toolbox macro 1 In the Toolbox Palette dialog box click Peptide Fragmentation Toolbox Figure 3 10 Peptide Fragmentation Setup 2 In the Setup tab ...

Page 406: ...lculation by clicking the peak in the list then clicking Delete Selected Peaks 4 If you will be identifying y and b pairs select the precursor peak then click Use Selected Peak Pairs To list ion pairs 1 Click the Pairs tab Figure 3 11 Peptide Fragmentation Pairs 2 Click the button that corresponds to the ion masses you want identified a and b pairs Lists peak pairs with a 28 Da mass difference a b...

Page 407: ...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 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 Sequence To list ion seq...

Page 408: ...ue the sequence search by clicking the Search Down or Up buttons The newly selected peak is now labeled as the reference peak and mass differences minus if you click Down or plus if you click Up are labeled if they correspond to an amino acid residue You can generate the complete sequence ladder by appending new results to existing results NOTE When you search using reference peaks selected from t...

Page 409: ... tab to another application such as Notepad or print as needed Correlation To list ion pair correlations 1 Click the Correlation tab 2 Select a peak in the Spec Peak List 3 Select the correlation you want to determine for the selected peak that is click a and b if you want to determine if the selected peak is a member of an ab fragment ion pair Figure 3 13 Peptide Fragmentation Correlation ...

Page 410: ...vely 5 Click Copy to Output Window Result Tab to copy results to the Result tab You can then copy from the Result tab to another application such as Notepad as needed 6 You can remove a peak from the Spec Peak list by selecting a correlation in the results list then clicking Delete Selected Peaks from Spec List Displaying the original labels To display the original labels select Peak Label from th...

Page 411: ...1 02 indicates a narrowly dispersed polymer A higher value for example 3 0 indicates a widely dispersed polymer Using Polymer Analysis 1 In the Toolbox Palette dialog box click Polymer Analysis Toolbox The range displayed in the Spectrum window is reflected in the X Display Range fields NOTE If you zoom on a different region in the Spectrum window the new range is not updated in the X Display Rang...

Page 412: ... It does not distinguish between different polymeric species that may be present in the mass range Enter an adduct mass Figure 3 14 Polymer Analysis Toolbox NOTE If the predominant ion series is M Na enter 23 for the adduct ion If the predominant ion series is M Ag enter 108 for the adduct ion If you are analyzing an unknown or a mixture leave this field blank the calculation is performed without ...

Page 413: ...more species are present Enter an adduct mass Enter the mass of the end group to subtract from the calculation You can leave this field blank if you do not know the end group mass CAUTION Correct labeling of the peaks is essential when using the labeled peaks option 4 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...

Page 414: ...ftware smooth the data perform a baseline correction then deisotope 2 If needed use Advanced Peak Detection parameters to adjust peak detection ranges and thresholds to screen out noise in low mass regions and to detect peaks in high mass regions 3 Set the Set the Base Peak Intensity or Max Peak Area in peak detection until the unwanted noise peaks are removed from the Spec Peak list Running MS Fi...

Page 415: ...the web site containing the database to search 4 Adjust settings on the web site as needed 5 Minimize the web page Do not exit 6 In the MS Tag dialog box enter Parent Ion mass if you are using MS Tag 7 Click Copy Peaks The Spec Peak list for the data file is copied to the web page 8 Start the search from the web site The database search is performed Results of the search are displayed on the web s...

Page 416: ...Appendix C Data Explorer Toolbox Visual Basic Macros C 20 Applied Biosystems C ...

Page 417: ...d replaced by Minimum Intensity 3 30 AC in spectrum header 2 31 5 34 Accelerating Voltage changes compensated for by system 5 25 effect on calibration 5 25 Accumulate spectra 4 22 Acquiring data comment displaying 1 15 comment displaying for open data file 1 15 comment displaying when opening a data file 2 3 version of software used for 1 15 Acquisition comment displaying for open data file 1 15 d...

Page 418: ...es to 1 34 importing traces from 1 35 Assign Macro 6 38 Auto Cal see Calibrating mass automatic Mariner data only Autocolor description 2 27 setting 1 26 Automatic Calibration On command dimmed 5 34 see also Calibrating mass automatic Mariner data only settings reference masses for Mariner Sequence Control Panel 5 27 settings reference masses for Voyager Sequence Control Panel 5 28 settings import...

Page 419: ...1 36 exporting from RSD and RCD 1 36 Binning data graphic compression 1 28 BO in chromatogram header 2 30 in spectrum header 2 31 5 46 BP Relative label on data cursor 1 27 BPI see also Base peak intensity in chromatogram header 2 30 in spectrum header 1 13 2 32 C CAL files see also Calibration constants applying 5 16 8 20 description 1 7 exporting during calibration 5 16 exporting from DAT 1 36 i...

Page 420: ...10 procedure 5 8 results 5 13 reverting to instrument calibration 5 22 single point calibration 5 8 troubleshooting 9 9 when to use 5 6 Calibrating mass PSD Voyager data only all segment masses considered 8 15 calibration reference file REF 8 21 constants applying A and B to new file 8 20 error message when selecting calibration reference file 8 14 internal calibration using known monoamino acid f...

Page 421: ...calibration 5 9 selecting for PSD calibration 8 13 viewing contents of 5 10 VOYAGER REF 5 18 Centering a peak 2 15 Centroid mass calculated during peak detection 3 69 calculating 3 39 copying from peak list 1 41 definition B 6 determining 5 36 improving accuracy of calculation during peak detection 3 26 labeling 3 57 Centroid Percent spectrum setting 3 26 Centroiding centroid data file size smalle...

Page 422: ...gram XAC from Mariner data creating from 4 13 extracted ion chromatogram XIC chromatogram creating from 4 5 improving signal to noise ratio 7 2 Mariner data displaying 1 12 2 2 Chromatogram window continued multispectrum Voyager data displaying 1 13 noise filtering 4 17 peak labels 3 52 peak list 3 38 results opening 2 40 retention time displaying 1 12 smoothing 4 17 spectrum number displaying 1 1...

Page 423: ... CNL Context sensitive menus 1 14 Converting see also Exporting DAT to ASCII 1 34 profile data to centroid 1 33 SPC to DAT 1 30 Voyager MS files 1 30 Copying all peaks 1 40 apex mass list 1 41 centroid mass list 1 41 data from other data files 2 37 data to Windows clipboard 1 38 displayed peaks 1 39 mass list 1 41 peak list 1 40 results 2 28 to metafile 1 38 trace data 1 39 2 37 trace image 1 38 C...

Page 424: ...playing retention time on 1 27 horizontal 1 27 labels 1 27 4 2 printing and suppressing 1 27 Data cursors continued turning on and off in graphic options 1 27 turning on and off in peak detection 3 23 vertical 1 27 Data Explorer moving between open files 2 8 starting and exiting the software 1 3 window customizing 1 17 window parts of 1 11 Data Explorer examples Mariner data 7 2 Voyager data 7 11 ...

Page 425: ...il imported 6 43 not overwritten when new software installed 6 43 DBE definition 6 6 Decimal points number displayed 3 55 3 56 DECONV in spectrum header 2 32 5 40 5 41 Deconvolution see Mass deconvolution Default settings applying from SET file 2 4 DEFAULTBLACK SET 1 19 1 23 Defaults background color 1 23 colors Mariner 1 4 colors Voyager 1 4 peak detection 3 2 processing and graphics settings app...

Page 426: ...description 6 2 elements adding 6 9 fragment ions electron state for accurate results 6 5 fragment ions identifying 6 2 Elemental composition continued Isotope Match Score 6 6 Isotope Match Score not reported for fragment ion calculations 6 6 isotope min and max values ignored 6 8 6 11 limits setting 6 7 6 9 6 12 Periodic table 6 10 procedure 6 3 results 6 5 Elemental targeting description 6 31 Is...

Page 427: ... 7 2 7 5 7 10 improving signal to noise ratio 4 6 including only mass of interest 4 6 trace label 2 31 4 7 4 8 4 16 Extraction mode setting 4 7 4 11 F Failed to create empty document message 9 3 Failed to open chromatogram data message 9 4 File format converting SPC to DAT 1 30 File management 1 30 File name does not print 9 13 full name not displayed 1 14 File properties searching 1 32 viewing 1 ...

Page 428: ...ro C 9 Fragment spectrum PSD see Segment spectrum PSD FRM files for macros 6 43 FWHM 6 20 G Gaussian Fitting peak detection 3 26 Gaussian smoothing see Smoothing Global peak detection parameters description spectrum 3 22 overriding for individual detection ranges spectrum 3 30 setting spectrum 3 13 Graphic options accessing 1 24 default settings for white or dark background 1 19 1 23 extracting fr...

Page 429: ... 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 ISO in spectrum header 2 32 6 6 6 16 6 18 6 33 Isotope average mass labeling 3 10 7 11 description B 1 displaying theoretical 6 33 isotopic envelope B 5 list of common B 8 mono...

Page 430: ...Trace labels AC 5 34 added and subtracted spectra 4 22 AdvBC 5 53 BC 4 29 5 47 BO 4 28 5 46 Labels continued CNL 4 9 CT 5 36 DECONV 5 40 5 41 filtered trace 4 25 ISO 6 6 6 16 6 33 MC 5 13 NF 4 19 5 44 NR 4 19 5 44 retention time displayed 4 22 RSM 5 44 SC 5 60 SM 4 19 5 44 TR 5 57 XAC 4 14 4 15 XIC 4 7 Ladder Sequencing macro C 2 C 5 Landscape printer orientation lost when you exit 9 13 setting pe...

Page 431: ...riner data Chromatogram window displaying 2 2 DAD data diode array detector displaying 1 12 DAD spectrum displaying 5 2 Mariner data continued examples 7 2 in source CID labeling C 9 isotope resolution limits 3 53 mass accuracy affected by peak shape 5 7 5 47 peak detection strategy 3 6 Mariner Sequence Control Panel automatic calibration settings reference masses for 5 27 MARINER REF viewing mass...

Page 432: ... 8 4 optimum focus and resolution observed near this mass 8 4 updated when Precursor mass changed 8 24 Maximize trace window 2 9 Maximum Charge State description 3 27 set too low 3 33 Maximum Isotopes setting 3 27 setting too low 3 35 MC in spectrum header 2 6 2 32 5 13 5 15 5 17 5 22 5 23 7 17 8 18 Metafile copying to 1 39 Microsoft applications Excel 1 5 Microsoft Office 1 5 Visual Basic Editor ...

Page 433: ... 2 30 4 19 in spectrum header 2 32 5 44 Noise filtering chromatogram 4 17 spectrum 5 42 spectrum recommended setting 4 18 5 43 Noise Threshold chromatogram calculated automatically 3 21 3 68 in peak detection algorithm 3 68 setting locally spectrum 3 30 Noise screening out noise filtering command 4 17 5 42 subtracting spectra and creating extracted ion chromatogram Mariner data 7 8 truncate spectr...

Page 434: ...ea peak centroid see Centroid mass charge state see Charge state peak deisotoping see Deisotoping inserting 3 39 Peak bounds color changing 1 25 in peak labels 3 55 3 58 Peak detection see also Detection Ranges see also Peak detection parameters see also Peak detection Mariner data see also Peak detection resolution based see also Peak detection Voyager data accessing 3 11 3 13 Advanced Settings t...

Page 435: ...efault settings 3 71 noise detected as peaks 3 7 strategy for 3 6 troubleshooting 3 6 Peak detection resolution based see also Peak detection default resolution 3 6 3 24 description 3 3 enabling 3 23 Filter Width value used 3 3 formula used to calculate number of data points 3 4 overriding 3 10 3 14 Peak detection Voyager data see also Peak detection complex digests 7 18 default settings 3 71 deis...

Page 436: ...9 parameters used 3 53 requirements 3 53 selecting 3 58 user labels 3 62 Peak labels filtering charge state 3 43 monoisotopic peak 3 43 Peak labels Mass Difference From Adjacent Peak regular labels 3 57 From Adjacent Peak user labels 3 62 From Selected Peak 3 57 Peak list charge state displaying selected 3 42 Chro and Spec 3 40 contents 3 38 copying apex masses only 1 41 copying centroid masses on...

Page 437: ...cape orientation 2 35 multiple files 2 34 peak list 3 44 traces 2 33 traces do not print 2 34 troubleshooting 9 13 without previewing 2 34 Process menu commands not displayed 9 8 Processing see also Processing settings commands not displayed on menu 9 6 troubleshooting 9 6 Processing History description 2 22 disabling 2 23 options 2 23 using 2 22 Processing settings applying when opening data file...

Page 438: ...2 38 2 39 2 41 opening 2 39 2 40 saving 2 40 Read only files viewing 2 7 Realign UV trace 4 30 Reapplying instrument calibration 5 22 Recording a macro 6 37 REF files see Calibration reference file REF Reference masses calibration see also Calibration reference file REF adding to REF file 5 18 displaying list of 5 10 8 16 for Voyager Sequence Control Panel 5 27 5 28 selecting 5 10 5 14 7 17 8 15 8...

Page 439: ...s 2 40 Retention time displaying for chromatogram 1 12 on data cursor 1 27 Reverting to instrument calibration 5 22 Right axis turning off 2 12 RNA residues labeling C 5 RSD file deleting 2 39 description 1 10 exporting 2 39 extracting information from 1 36 opening 2 39 RSM in spectrum header 2 32 5 44 RST files see also Results name of raw data file result is derived from 2 38 2 39 2 41 not affec...

Page 440: ...tensity summing for one mass 5 2 Signal to noise ratio algorithm 6 23 calculating 6 23 improving 5 2 7 2 7 3 Single charge conversion description 5 59 example 5 61 multiply charged peaks seen after conversion 5 62 procedure 5 59 troubleshooting 5 62 Single point calibration see Calibrating mass SM in chromatogram header 2 30 4 19 in spectrum header 2 32 5 44 Smoothing chromatogram 4 17 setting met...

Page 441: ...d ion chromatogram XIC creating from 4 5 4 8 linking in different data files 2 13 Mariner DAD data displaying 1 13 mass deconvolution 5 37 noise filtering 5 42 Spectrum window continued organizing 2 13 peak list 3 38 peak centering 2 15 resolution mass calculating 6 20 results opening 2 40 signal to noise ratio calculating 6 23 smoothing 5 42 spectrum numbers on trace do not match axis 9 5 subtrac...

Page 442: ...6 ToolTips 1 12 TR in spectrum header 2 32 5 57 Trace browser 2 9 Trace labels cannot be removed 2 30 chromatogram 2 30 spectrum 2 31 Trace windows maximizing 2 9 Traces see also Trace labels see also Traces copying see also Traces removing adding 2 16 2 18 annotating 2 28 axes setting 2 11 bar mode 1 28 centering a peak 2 15 centroid creating 5 36 changing colors to black before printing 2 33 col...

Page 443: ...ignored 6 8 6 11 error messages when you open a file 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 peak detection 9 14 peak detection Voyager 3 9 peak labeling 9 14 printer does not stay set...

Page 444: ...data for C 3 References required C 3 Voyager MS files converting to DAT not supported 1 30 Voyager data Chromatogram window displaying 1 13 2 7 examples 7 11 isotope resolution limits 3 53 peak detection strategy 3 8 Voyager Sequence Control Panel automatic calibration settings reference masses for 5 28 VOYAGER REF provided 5 18 viewing masses in 5 10 VOYAGERPSD SET 8 11 W Warranty exceptions A 3 ...

Page 445: ...2 11 XIC see also Extracted ion chromatogram XIC in chromatogram header 2 31 Y y and b ion pairs labeling C 11 Y cursor setting 1 27 Y axis offsetting chromatogram 4 27 offsetting spectrum 5 45 scaling 2 12 spectrum 2 11 Z z labels 3 58 z average molecular weight determining C 15 Zero charge state displayed in peak list 3 40 3 43 Zero charge spectrum for overlapping peaks 5 40 for resolved peaks 5...

Page 446: ...I D E X N Index 30 Applied Biosystems ...

Page 447: ...sonnel reaches into 150 countries on six continents For sales office locations and technical support please call our local office or refer to our web site at www appliedbiosystems com Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses www appliedbiosys...

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