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Data Explor er ™ Softwar e V ersion 4 Series Softwar e User Guide.
© Copyright 200 1, Applied Bi osyst ems . All right s reserve d. For Research Use On ly. Not for use in diag nosti c procedures. Informat ion in this do cument i s subject to c hange wit hout notice. A pplied Biosy stems assume s no responsib ility for any errors t hat may appea r in this docume nt.
Table of C onten ts Data Explorer ™ Softwar e Us er ’ s Guide i ii Table of Contents How to Use This Guide ........... ................... ................... ............. xi Chapter 1 Data Explorer™ Basics 1.1 Overview ........... ............
Table of Contents iv Applied Bios ys te ms Chapter 2 U sing Chromatog ram and Sp ectrum Windows 2.1 Opening and Closing Dat a Files ................... ................... ........... 2-2 2.1.1 Opening Data Files ................ .....................
Table of C onten ts Data Explo rer ™ Softw ar e User ’ s Guide v Chapter 3 Peak Detection and Labeling 3.1 Overview ........... .............. .................. ................... ................ 3-2 3.1.1 Default Peak Detection ...............
Table of Contents vi Applied Bios ys te ms Chapter 4 Examining Chromatogram Dat a 4.1 Overview ........... .............. .................. ................... ................ 4-2 4.2 Creating an Extracted Ion Chromatogram ....... .............. ...
Table of C onten ts Data Explo rer ™ Softw ar e User ’ s Guide vi i 5.5 Centroiding ............ .............. ................... ................... .......... 5-36 5.6 Mass Deconvolution (Mariner Dat a Only) ... ................... ...........
Table of Contents viii Applied Bios ys te ms Chapter 7 D at a Explorer Ex amples 7.1 Mariner Dat a Examples ................ ................... .................. ....... 7-2 7.1.1 Improving Signal-T o-Noise Ratio ........................ ...........
Table of C onten ts Data Explo rer ™ Softw ar e User ’ s Guide i x Appendix A W arranty Information ............... .............. ........... A-1 Append ix B Overview of Isotopes ............... .................. ....... B-1 Append ix C Data Explorer T ool box (Visual Basic Macros) .
Table of Contents x Appli ed Bios ys te ms.
How t o Use T his Gu ide Data Explorer ™ Softwar e User ’ s Guide xi 1 How to Use Th is Guide Purpose of this guide Th e Applied Biosystems Dat a Ex plorer ™ S oftware User ’ s Guide describes proce ssing and a nalyzing d ata with the Data Explo rer software.
How to Use This Gu i de xii Applied Bi osystems 1 Chapter 5, Examining Spectrum Data Describes processing and analyzing mass spectral data. Chapter 6, Using T ools and Applications Describes how to ge.
How t o Use T his Gu ide Data Explorer ™ Softwar e User ’ s Guide xii i 1 Conventions Th is guide us es the follow ing conv entions t o make tex t easier to understand. • Bold indi cates user ac tion. For example: T ype 0 and press Ente r for the remaining fields.
How to Use This Gu i de xiv Applied Bi osystems 1 Related docu mentat ion The relate d documen ts shipped wi th your system inc lude: • Mariner ™ Workstation User ’ s Guide — Us e this document to learn detail ed informati on about the Mariner Works tation.
1 Chapter Data Explorer ™ So ftw are User ’ s Guide 1 -1 1 Dat a Explorer ™ Basics This chapter contains the following sections: 1.1 Overview ......... ....................... ................... 1-2 1.2 File Formats and T ypes ............... .
Chapter 1 Data Explor er ™ Basics 1- 2 Applied Biosystems 1 1.1 Overview Description The Da ta Explorer ™ V er sion 4.0 pr ocessing sof tware is graphic al software that you us e to anal yze, calib rate, and report data.
Overview Data Explorer ™ So ftw are User ’ s Guide 1 -3 1 S tarting and exiting the software T o s tart the Data Explore r software f rom the Win dows NT deskto p, double- click th e Dat a Explorer icon on th e desktop . The Data Expl orer windo w opens.
Chapter 1 Data Explor er ™ Basics 1- 4 Applied Biosystems 1 Figure 1-2 Data Explorer Window wi th Voyager Dat a Default co lors The defau lt colors are d ifferent for Mar iner and V oyage r: • Mar.
File Formats a nd Typ es Data Explorer ™ So ftw are User ’ s Guide 1 -5 1 1.2 File F ormats and Type s This sect ion descr ibes: • Soft ware ap plicati ons comp atib ility • Data (.
Chapter 1 Data Explor er ™ Basics 1- 6 Applied Biosystems 1 Mariner .SPC file format In Mariner so ftware version s earlier than v ersion 3.0, data files are stored in .SPC fo rmat. Y ou can vi ew and pr ocess .SP C files in Data Explorer , or you can co nvert these fil es to .
File Formats a nd Typ es Data Explorer ™ So ftw are User ’ s Guide 1 -7 1 Settings (continued) .MSM (Mariner only) MS Method settings, if dat a was acquired using an .MS M f i l e. NOTE : T o access the instrument settings used to acquire each spectrum in an MS Method, you must first extract the .
Chapter 1 Data Explor er ™ Basics 1- 8 Applied Biosystems 1 Additional files types Additi onal file ty pes you may see on y our sys tem are described below . T able 1 -2 Additional File T ypes Catego ry File T ype File Co ntent Data .PKT T ext file containing a chromatogram or a spectrum peak list that you can save from the Output window .
File Formats a nd Typ es Data Explorer ™ So ftw are User ’ s Guide 1 -9 1 Reference .REF List of masses to select from during calibration. See “ Creating and saving a calibration reference file ” on page 5-18. Process .RCT (Mariner only) Results file saved from: • Mariner .
Chapter 1 Data Explor er ™ Basics 1- 10 Applied Biosystems 1 Process (continued) .RCD Chromatogram result s file exported from .DA T files. See Section 2.7, Export ing, Opening, and Deleting .RCD and .RSD Resul ts Files ( Mariner Data Only ). .RSD Spectrum results file exported from .
Parts of the Data Ex plorer Window Data Explorer ™ So ftw are User ’ s Guide 1 -1 1 1 1.3 Parts of the Data Expl orer Window This sect ion descr ibes: • Overvie w • T ool bar • Chromato gram.
Chapter 1 Data Explor er ™ Basics 1- 12 Applied Biosystems 1 To o l b a r The to olbar contains buttons that access Data Explorer functions. For a desc riptio n of a toolb ar button, pl ace the cur sor on the button. A br ief descrip tion of the button (T ool T ip) is di splayed below the b utton.
Parts of the Data Ex plorer Window Data Explorer ™ So ftw are User ’ s Guide 1-1 3 1 SPEC Displays the spectr um for the selected time in the TIC or T AC trace. By default, displays spectrum #1. The trace label includes “ DAD ” for spectra selected from T AC.
Chapter 1 Data Explor er ™ Basics 1- 14 Applied Biosystems 1 Labels in the chromatogram or spectrum title identify t he type of data displayed in the window .
Parts of the Data Ex plorer Window Data Explorer ™ So ftw are User ’ s Guide 1-1 5 1 Output window The O utput window ( see Figure 1-3 on page 1- 1 1) displays tabs at the b ottom that y ou can cl.
Chapter 1 Data Explor er ™ Basics 1- 16 Applied Biosystems 1 • Instrument Setting — Di splays a list of inst rumen t setting s used to obtain th e data.
Customizing the Data Explorer Window Data Explorer ™ So ftw are User ’ s Guide 1-1 7 1 1.4 Cust omizing the Data Ex plorer Win dow This sect ion incl udes: • Setting d efault value s • Customiz ing Graph ic and P rocessing s ettings • Customiz ing toolb ars 1.
Chapter 1 Data Explor er ™ Basics 1- 18 Applied Biosystems 1 Overvi ew of processing and graphic settings Processi ng and grap hic settin gs control ho w data is processed and displ ayed in the Data Explore r software. The last s ettings used are au tomatica lly saved in the data file when you c lose it.
Customizing the Data Explorer Window Data Explorer ™ So ftw are User ’ s Guide 1-1 9 1 Additi onal .SET file s that have been dev eloped for detec tion of different types of data are inc luded in the C:VOY AG ERPROGRA MSET FILE S direct ory . The names of the .
Chapter 1 Data Explor er ™ Basics 1- 20 Applied Biosystems 1 Opening, customizing, and saving .SET file s T o ope n, custom ize, and save .SE T files: 1. If you are customizing a default .SET file, make a copy of the original file befo re opening it.
Customizing the Data Explorer Window Data Explorer ™ So ftw are User ’ s Guide 1-2 1 1 T o use th e Setti ngs opt i on: 1. Select Settings from the File menu.
Chapter 1 Data Explor er ™ Basics 1- 22 Applied Biosystems 1 4. T o re move a button from a too lbar , c lick-drag the button from the to olbar . NOTE : The Customize dialog box must be displayed to click-drag a button from a toolbar . 5. Cli ck OK to close t he Cust omize dialog b ox.
Setting Grap hic Optio ns Data Explorer ™ So ftw are User ’ s Guide 1-2 3 1 1.5 Setting Graphic Options This sect ion incl udes: • Changing bac k ground colo r • Customiz ing options • Reverting t o previo us graphic options NOTE: Changes you make to Graphic Options are saved with the data file.
Chapter 1 Data Explor er ™ Basics 1- 24 Applied Biosystems 1 1.5.2 Customizing Graphic Options This se ct ion inclu de s: • Acces sing gr aph ic option s • Settin g colors • Setting l ine widt.
Setting Grap hic Optio ns Data Explorer ™ So ftw are User ’ s Guide 1-2 5 1 Figure 1-4 Graphic Op t ions Dialog Box — Graph Setup T ab Setting colors Y ou can s et color s manua lly or auto maticall y .
Chapter 1 Data Explor er ™ Basics 1- 26 Applied Biosystems 1 When you manually set colors , note: • Selecti ons set to wh ite (or li ne widths set to 0 ) may not print on certain pr inters. • If you sel ect different trace colors for multiple tr aces, only the color for the active tr ace is save d when you close th e data file.
Setting Grap hic Optio ns Data Explorer ™ So ftw are User ’ s Guide 1-2 7 1 Setting data cursors T o e nable data cu rsors an d set curs or labels and attrib utes: 1.
Chapter 1 Data Explor er ™ Basics 1- 28 Applied Biosystems 1 Setting traces in Line or V ertical Bar mode Y ou can change th e trace display from Line to V ertic al Bars. Each vertical bar repre sents one data po int. V er tical bar mod e is useful when setting peak detecti on parameters to determine th e number of points across a peak.
Setting Grap hic Optio ns Data Explorer ™ So ftw are User ’ s Guide 1-2 9 1 1.5.3 Reverting to Previous Graphic Options Y o u have two options to r evert to pr eviously used graphi c options : • Revert to Last Saved Gra phic Set ting — Reverts to the las t graphic s ettin gs save d in the data file.
Chapter 1 Data Explor er ™ Basics 1- 30 Applied Biosystems 1 1.6 Ma naging Fil es This se ction desc ribes: • Conver ting .SPC fil e format to .DA T file f ormat (Mar iner only) • Conver ting da.
Managing Files Data Explorer ™ So ftw are User ’ s Guide 1-3 1 1 Before you begin Co nf ir m t ha t th e .SPC and .CGM files are located in the same directory . Use Windows NT ® Explorer to display the directory contents and to move the .SPC and .
Chapter 1 Data Explor er ™ Basics 1- 32 Applied Biosystems 1 The Convert to .DA T Format dialog box reappears. 5. Cli ck OK . A message box is displayed, showing the file name of the newly created .DA T file. 6. Close the .SPC file an d open the . DA T file before processi ng.
Managing Files Data Explorer ™ So ftw are User ’ s Guide 1-3 3 1 1.6.2 Converting Dat a from Profile to Centroid (Mariner Dat a Only) Overview Y o u can conve rt an entire data file from p rofile to c entroid format. Centroid format files are sm aller than p rofile fo rmat file s.
Chapter 1 Data Explor er ™ Basics 1- 34 Applied Biosystems 1 1.6.3 Converting to and Exporting ASCII D at a This se ction desc ribes: • Conver ting a data file t o ASCII forma t • Exporti ng a t.
Managing Files Data Explorer ™ So ftw are User ’ s Guide 1-3 5 1 1.6.4 Importing a T race in ASCII Format Y o u can import tr ace data in ASC II format.
Chapter 1 Data Explor er ™ Basics 1- 36 Applied Biosystems 1 CAUTION An imported ASCII format trace contains only the dat a points for the trace. The Sample Info and Instrument settings tabs in the Output window display data from the data file yo u opened in step 1.
Managing Files Data Explorer ™ So ftw are User ’ s Guide 1-3 7 1 NOTE : T o export calibration constants used t o acquire the dat a, selec t Mass Calibration from the Process menu, then select the Revert to Instrument Calibration before exporting.
Chapter 1 Data Explor er ™ Basics 1- 38 Applied Biosystems 1 Saving .LBS and . LBC files T o save s pectrum (. LBS) or chroma togram (. LBC) peak label files from a .DA T , .RSD, o r .RSC file, s ee Section 3.5.3, Setting Cu stom Pea k Labels . 1.6.
Managing Files Data Explorer ™ So ftw are User ’ s Guide 1-3 9 1 NOTE: If you paste the image into an application that does not handle Windows Metafile format images, for example Microsoft Paint, images are distorted. Copy trace data T o c opy ra w data (x,y pairs) f or the peaks displa yed in the active tr ace to the W indows clipboa rd: 1.
Chapter 1 Data Explor er ™ Basics 1- 40 Applied Biosystems 1 NOTE: Copy Display ed Peaks copies all fields and headings. However , some data applications may not work correctly if headings are present because the first row contains text and not dat a.
Managing Files Data Explorer ™ So ftw are User ’ s Guide 1-4 1 1 2. Se le ct t he trace window to copy . 3. Di splay the peak lis t by selec ting Output W i ndow fr om the Dis play menu , then clic king the C hro Peak List or Spec Peak List ta b. 4.
Chapter 1 Data Explor er ™ Basics 1- 42 Applied Biosystems 1.
2 Chapter Data Explorer ™ So ftw are User ’ s Guide 2 -1 2 Using Chromatogram and Spect rum Windows This chapter contains the following sections: 2.1 Opening and C losing Data Files .................... ..................... 2-2 2.2 Adjusting the Display Range .
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 2 Applied Biosystems 2 2.1 Open ing and Closin g Data Files This se ction incl udes: • Open ing dat a f i les • Displa ying Mar iner UV trac es • Disp la yi ng V oya ger chr om a tog ra m s • Viewing read- only files • Moving between ope n files • Closin g data files 2.
Opening and C losing Data Fil es Data Explorer ™ So ftw are User ’ s Guide 2 -3 2 . Figure 2-1 Select Files D ialog Box 2. Click the down arrow to display the Files of type list, then select the file extension to display . 3. Select up to eight data f iles to open, then click Ad d or Add All .
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 4 Applied Biosystems 2 Specifying settin gs 5. Select a Restoring G raphics and Proces sing Setti ngs option to a pply to ne w files you are ope.
Opening and C losing Data Fil es Data Explorer ™ So ftw are User ’ s Guide 2 -5 2 Figu r e 2-2 Data Explo rer Window with Four Mar iner Data Files Open (Each .
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 6 Applied Biosystems 2 The data is lab eled acc ordingly: Auto matic ally ru nnin g macr os Y ou can set macr os to aut omatically run when you open o r close fi les. For informat ion, see “ Ru nning Macros Automa tically When Opening and Closi ng Files ” on page 6-45.
Opening and C losing Data Fil es Data Explorer ™ So ftw are User ’ s Guide 2 -7 2 2.1.3 Displaying V oyager Chromatograms T o display c hromatogra ms for multis pectrum V o yager .DA T fi les: 1. Open the .DA T files as desc ribed in “ Opening Data Files ” on page 2-2.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 8 Applied Biosystems 2 2.1.5 Moving Between Open Files Y ou ca n have more than on e file open at a time .
Opening and C losing Data Fil es Data Explorer ™ So ftw are User ’ s Guide 2 -9 2 Figure 2-5 Select Fi le to Activate Dialog Box 3. S el e ct : • Maximize — T o maximize the Spectrum window of.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 10 Applied Biosystems 2 2.1.6 Closing Dat a Files Y ou can close fi les in t he followi ng ways: • Selec t Close from the File menu to clos e the active file. • Selec t Close All Files from the File menu to c lose all files.
Adjusting the D isplay Rang e Data Explorer ™ So ftw are User ’ s Guide 2-1 1 2 2.2 Adjusting the Display Range T o s et the dis play rang e: 1. Click the Chromatogram or Spectrum window to activate it. 2. F rom the Dis play me nu, select Range . 3.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 12 Applied Biosystems 2 5. Set the parameter s describe d below as needed: 6. Cli ck OK . Parameter Descr iption Sca ling M ode Display Relative Autoscales the trace to the l argest peak in the selected range.
Organizing Window s Data Explorer ™ So ftw are User ’ s Guide 2-1 3 2 2.3 Organizing Windows Linking views Linking C hromatogram or Spectrum windows i n different data file s allows you to zoom on multiple data files. NOTE: When different data files are linked, zooming functions performed on one data file ar e applied to all linked files.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 14 Applied Biosystems 2 2.4 Manipulating Traces This se ction incl udes: • Zoomi ng centerin g, and cus tomizin g a trace • Duplic ating a t.
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-1 5 2 Centering a peak in the trace T o cen ter a peak in th e trace w indow: 1. Display the trace containing the peak of i nterest. 2. Click t he Spec Peak List or Chr o Pe ak List tab in the Output wind ow.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 16 Applied Biosystems 2 For example, if y ou select Divide Active T race to Four when the active trace has a range of 0.
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-1 7 2 Wh en you pe rform c ertai n func tions (for ex ample, smoothing ), a new trac e is crea ted.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 18 Applied Biosystems 2 2. Sel ect the Re place Mode: • Replace the Acti ve T race (default) — Replaces the a ctive trace wi th the n ewly cr eated t race. • Add a New T race — Adds the newly created trace to the window .
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-1 9 2 Figure 2-8 Adding T races (Four Traces Shown, u p to Four More Can Be Added) When y ou perform a function that adds a ne w trace, the label of the tr ace changes from Not Us ed to the la bel for the t ype of trace created ( Fi gure 2-9).
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 20 Applied Biosystems 2 Figure 2-9 shows the original tr ace and t hree added tr aces that now contain a smooth ed spect rum (SM), a centroided spectru m (CT), and a baseline offset spectrum (BO).
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-2 1 2 2.4.5 Removing T races Removing the active trace T o re move the active trace from a window: 1. Cl ick the trace to remove. 2. C lick in the toolba r , or r ight-cli ck the trac e, then select Remove T race from the menu.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 22 Applied Biosystems 2 2.4.7 Recalling and Rearranging T races (Processing History) Overvi ew The Ch romatogram and Spec trum windows can display up to 8 traces at a time for a d ata file. However , up to 16 traces are held in m emory .
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-2 3 2 Sett ing Processing History options T o set Proce ssing Hi story o ptions: 1. From the T ools menu, select Processing History Optio ns . The Processing History Options dialog box (Figure 2-10) is di splayed.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 24 Applied Biosystems 2 2.4.8 Overlaying T races This se ction incl udes: • Overla ying tra ces from di fferent data files • Overla ying tra.
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-2 5 2 NOTE: When saving results, only the results for the active trace are saved. Over laying traces in a single data file T o o verlay trac es in a si ngle data file: 1. Display the chromatogram or spectr um traces you want to overlay .
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 26 Applied Biosystems 2 Changing the active trace T o ch ange the ac tive trac e in an over lay: 1. From the Display menu, deselect Overlay . 2. Cli ck the trace t o activa te. 3. From the Display menu, se le ct Overla y .
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-2 7 2 NOTE: Y ou must select the Use same settings for all traces check box before selecting options for traces.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 28 Applied Biosystems 2 2.4.9 Annot ating T races Y ou can add text a nnotation to tr aces by: • Copyin g a line of r esults from the Output w.
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-2 9 2 Annotating the trace T o annotate the trace: 1. Click the trace at the location where you want to insert text. 2. R ight-clic k, then s elect: • Paste text — If you copied results.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 30 Applied Biosystems 2 2.4.10 V iewing T race Labels The Data Explo rer so ftware incl udes a label in the tr ace header to identify the type of da ta displaye d. NOTE: T race labels ar e applied by the software and cannot be removed.
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-3 1 2 Figure 2-1 1 illustrates an extracted ion chroma togram with a “ XIC ” chromatogram trace labe l.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 32 Applied Biosystems 2 BPI Base peak i ntensity CT Centroid DAD Diode array data DECONV (Mariner dat a only ) Zero char ge dec onvoluted trace .
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-3 3 2 Figure 2-12 illus trates a sm oothed sp ectrum with an “ SM5 ” spectrum trace labe l. Figure 2-12 Spectrum Trace L abel 2.4.1 1 Printing T races Printing traces T o print traces: 1.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 34 Applied Biosystems 2 3. T o pri nt with the x -axis along the longes t length of th e pa per , select Pr int Setup from the Fi le menu, then select Landscape orientation. NOTE: T he Landscape printing orientation you set in Data Explorer is lost when you close Dat a Ex plorer .
Manipulating Tra ces Data Explorer ™ So ftw are User ’ s Guide 2-3 5 2 NOTE: Line Widths of 0 or 1 (or lines set to the color white) may not print on ce rt ai n printers. If tr aces do not print, change the line wid th (or color). Dedicating a printer to landscape orientation T o d edicate t he printer to landscap e orientation: 1.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 36 Applied Biosystems 2 2.5 Working with Multiple Data Fi les When yo u have multiple data files open, you can: • Work with the d ata files separately to view , zoom , and prin t • Copy tr aces from one d ata file to anoth er to compare or combi ne data 2.
Working with Multiple Data Files Data Explorer ™ So ftw are User ’ s Guide 2-3 7 2 NOTE : If you select Print All V iews when more than two dat a files are open, certain printers may not print the dat a file name.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 38 Applied Biosystems 2 Comparing copied tr aces Af ter you cop y a trace to another tra ce window , you can compare tr aces by over laying (see Secti on 2.4. 8, Overla ying T races) or by using trace arithmet ic (see Secti on 5.
Exporting, Opening, and Deleting .RCD and .RSD Results Files (Mariner Data Only) Data Explorer ™ So ftw are User ’ s Guide 2-3 9 2 2.7 Ex porting, Open ing, and Deleting .RCD and .RSD Results Files (Ma riner Data Only) Exporting results for .RCD and .
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 40 Applied Biosystems 2 Deleting resu lts for .RCD and .RSD files Use Windows NT Explorer to delete .RC D and .RSD res ult files. 2.8 Saving, Op ening, and Dele ting .SPC Results Files (Mariner Data Only) Savi ng results for .
Saving, Opening , an d Deletin g .S PC R esults Files (Mariner Data Only) Data Explorer ™ So ftw are User ’ s Guide 2-4 1 2 3. S elect the .RS T or .RCT file to open, then cl ick OK . NOTE: Saturation Correction is not applied to Mariner .RST files.
Chapter 2 Using C hro matogr am and Spectrum Windows 2- 42 Applied Biosystems 2.
3 Chapter Data Explorer ™ So ftw are User ’ s Guide 3 -1 3 Peak Detection and Labeling This chapter contains the following sections: 3.1 Overview ...................... ................. .................. 3-2 3.2 Peak Detection ............. ....
Chapter 3 Pe ak Detecti on and Labeling 3- 2 Applied Biosystems 3 3.1 Ov erview This se ction incl udes: • Default pe ak detectio n • The re solution -based peak detec tion rout ine 3.1.1 Default Peak Detection Overv iew When you open a data file, it i s automa tically pe ak detect ed.
Overview Data Explorer ™ So ftw are User ’ s Guide 3 -3 3 3.1.2 The Resolution-Based Peak Detection Routine This sect ion descr ibes: • T y pe of data affected • Proces s that occu rs • Dete.
Chapter 3 Pe ak Detecti on and Labeling 3- 4 Applied Biosystems 3 The sof tware uses the followi ng formula t o calculate the expecte d number of da ta points in a pea k: Detection ranges Figure 3- 1 is an example of the resol ution-based detectio n ranges automatic ally calc ulated by the softwa re.
Overview Data Explorer ™ So ftw are User ’ s Guide 3 -5 3 Overla ppin g peak detect ion rang es T o accommo date spe ctral peaks that occur on the bo undary of two pe ak detectio n ranges , the softwa re creates de tection ranges th at overlap (Fi gure 3-2 ).
Chapter 3 Pe ak Detecti on and Labeling 3- 6 Applied Biosystems 3 3.2 Peak Dete ction This se ction incl udes: • S trateg y for Mariner peak detec tion • S trategy f or V oy ager peak d etection • Setting peak detec tion parameter s • Peak de tection paramet er descripti ons • Charge state determination and examp les 3.
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3 -7 3 3. I f peak dete ction is not acceptable , leave th e Use Reso lution Dep endent Set tings opti on enable d, and adjust the follow .
Chapter 3 Pe ak Detecti on and Labeling 3- 8 Applied Biosystems 3 3.2.2 Strategy for V oyager Peak Detection This se ction give s some quic k suggesti ons on how to approac h V o yager peak detection . For details on peak detectio n, see Secti on 3.2.
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3 -9 3 • Deisot ope (reflect or dat a only) — Peak deisotoping reduces the spectrum to a monoisotopic cen tr oid ed plo t o f t he m on ois ot op ic masses. This is us eful in ident ifying ov erlapping isotope c lusters.
Chapter 3 Pe ak Detecti on and Labeling 3- 10 Applied Biosystems 3 4. If you see more th an one of the proble ms listed above i n a spectrum, you can adjust peak detection parameters for any or a ll d.
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-1 1 3 3.2.3 Setting Peak Detection Parameters This section incl udes: • Before y ou begin • Settin g chromatogra m parameters • Se.
Chapter 3 Pe ak Detecti on and Labeling 3- 12 Applied Biosystems 3 Figure 3-3 Chromatog ram Peak Detection Setup Dialog Box 4. Se lect a de tection r ange and set parameters as needed. 5. T o ap ply setti ngs to al l traces, select Us e same settings for all t races in view .
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-1 3 3 Setting Basic Settings (spectrum data) Basic S ettings sho uld prov ide acceptable peak detection for most ap plications . After you apply the se parameters, no further adjustm ent shoul d be requ ired.
Chapter 3 Pe ak Detecti on and Labeling 3- 14 Applied Biosystems 3 Figure 3-4 Sp ectrum Peak Det ection Setup — Basic Settings T ab 5. If y ou are detec ting PSD data, or want to over ride the Global Thresholds , select Use Advanced Settin gs and skip to st ep 7.
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-1 5 3 NOTE: Resolution-dependent settings do not apply to Mariner chromatogram data or Voyager PSD dat a. For more infor mation, see S ection 3.1.2, The Resolution-Based Peak Detection Routine.
Chapter 3 Pe ak Detecti on and Labeling 3- 16 Applied Biosystems 3 Setting Peak Processing parameters (spectrum data only) T o s et Peak Pr ocessing parame ters: 1. Click the Peak P rocessing tab in the S pe ctr um Peak Detection Setup dialog box. The Peak Processing tab is displayed (Figure 3-5).
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-1 7 3 Setting Advanced Settings (spectrum data only) T o set Adva nced Setti ngs that y ou can a pply local ly to a selec ted detection range, an d that overr ide the thre sholds set in the B asic Settings tab: 1.
Chapter 3 Pe ak Detecti on and Labeling 3- 18 Applied Biosystems 3 3. Se lect a de tection r ange, then set paramete rs as neede d. 4. Cli ck App ly to accept the parameters an d leave the dialog box open , or click OK to ac cept the para meters and close the dialo g box.
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-1 9 3 3.2.4 Peak Detection Parameter Descriptions This sect ion descr ibes: • Chromatog ram settings • Basic S ettings (s pectrum da.
Chapter 3 Pe ak Detecti on and Labeling 3- 20 Applied Biosystems 3 Detection Ranges (continued) T o delete a range, select the range, then click . T o combine all ranges in the list into one range, click . The peak detection settings displayed in the dialog box correspond to the selected range.
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-2 1 3 NOTE: In previous versions of Data Explorer software, peak detection allowed you to specify Peak Wid th and Noise Threshold for chromatogram data.
Chapter 3 Pe ak Detecti on and Labeling 3- 22 Applied Biosystems 3 Basic S ettings (spectrum data only) T abl e 3-2 describes the parameters i n the Basic S ettings tab of the Spe ctrum Peak Detection Setup di alog box (see Figure 3-4 on page 3-14). Default pea k detecti on value s are list ed in Sec tion 3.
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-2 3 3 %Max Peak Area Specifies a percentage of the peak with the largest area as the threshold value. T o be detected, peaks must be above this threshold and above the %Base Peak Intensity value.
Chapter 3 Pe ak Detecti on and Labeling 3- 24 Applied Biosystems 3 Peak Resolution Mass Resolution V alue used to determine the Filter Width used for detection.
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-2 5 3 NOTE: In previous versions of Data Explorer software, peak detection allowed you to specify Peak Wid th. The software now automatically uses a minimum peak width that is equal to the Filter W idth and a maximum peak wid th of 10,000 data point s.
Chapter 3 Pe ak Detecti on and Labeling 3- 26 Applied Biosystems 3 Peak Proc essing parameters (spectrum data only) T abl e 3-3 describes the parameters i n the Peak P rocessing tab of the Spec trum Peak Detection Setup d ialog box (s ee Figure 3-5 on page 3-16).
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-2 7 3 Charge St ate Determination (Spectrum only) Maximum Charge St a t e Determines the peak spacing evaluated for the presence of isotope peaks. Th e ex pe ct ed p ea k s pa ci ng i s d ete rm i ned by the Max Ch arge S tate plus or minus a tolerance value.
Chapter 3 Pe ak Detecti on and Labeling 3- 28 Applied Biosystems 3 Adva nced Settings (spectrum data only) T abl e 3-4 describes th e parameters in th e Advanced Settings tab of the Spec trum Peak Detection Setup dia log box (see Figure 3-6 on page 3-17) .
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-2 9 3 Detection Ranges (continued) Y ou can set multiple, non-contiguous ranges and define parameters for each range independently . Y ou select a range in the Detection Ranges list box by single-clicking the range number .
Chapter 3 Pe ak Detecti on and Labeling 3- 30 Applied Biosystems 3 Active Range Thresholds NOT E: These settings apply to the Detection Range selected, and override the Global Thresholds specified on the Basic Settings tab (described on page 3-19). %BP Intensity See “ %Bas e P eak Intensity ” on page 3- 20.
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-3 1 3 Filter S ettings Width Number of data points used i n smoothing for peak detection before integration. This value is automatically calculated by the software if you select Use Resolution Dependent Settings on the Basic Settings tab.
Chapter 3 Pe ak Detecti on and Labeling 3- 32 Applied Biosystems 3 3.2.5 Char ge S t ate Determination and E xamples NOTE: Isotope-resolv ed peaks in V oyager data are typically singly charged. See Section 3.7, Default Peak Detection Settings, for recommended settings.
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-3 3 3 Max Cha rge State and Max Isotope # set correct l y When th e Max Char ge S tate and M ax Isotope # are set correc tly (in the example sh own in Figure 3-7, both ar e set to 4), the neurote nsin 558 m/z isotope cl uster contains fo ur peaks at charge state 3.
Chapter 3 Pe ak Detecti on and Labeling 3- 34 Applied Biosystems 3 . Figure 3-8 Max Charge S tate Set T o o Low and Max Isotope # Set Correctly The groupi ng of the fi rst and fou rth peaks i nto an isotop e cluster i s apparent w hen yo u turn on mono isotopic peak li st filtering ( Figure 3-9).
Peak Detectio n Data Explorer ™ So ftw are User ’ s Guide 3-3 5 3 Max Isotope # set too low I f the Max Is otope # is s et too low, the software incorrec tly groups pe aks into di f fere nt isotope c lusters.
Chapter 3 Pe ak Detecti on and Labeling 3- 36 Applied Biosystems 3 Effect of Minimum Intensity The Minimum I ntensity setting o n the Adva nced Set tings tab can also de termine how charge s tates are deter mined for a peak, because it determin es if the s oftware can find a matc h for a pea k.
Peak List Data Explorer ™ So ftw are User ’ s Guide 3-3 7 3 3.3 Peak List This sect ion descr ibes: • Display ing the p eak list • Inserti ng peaks in the pea k list • Saving the pea k list • Sortin g, filtering , and printin g the pe ak list 3.
Chapter 3 Pe ak Detecti on and Labeling 3- 38 Applied Biosystems 3 Contents of peak list The peak lists contain th e followi ng informati on. Chro matogram peak list Each ent ry repre sents one chro m.
Peak List Data Explorer ™ So ftw are User ’ s Guide 3-3 9 3 3.3.2 Inserting Peaks in the Peak List Descr iption If ch romatogram or spectrum peaks are not detecte d and labele d by the select ed d etection parame ters, you can manu ally detect and label peaks by ins erting p eaks in the peak l ist.
Chapter 3 Pe ak Detecti on and Labeling 3- 40 Applied Biosystems 3 Inserted peaks a re: • Removed from the list when you close the dat a file, reprocess the data, or s et peak detection parameters so th at the inser ted peak is no longer detected. • Assigned a charge state of 0 to indicate the charge state is unknown.
Peak List Data Explorer ™ So ftw are User ’ s Guide 3-4 1 3 NOTE: Inserted peaks are included when you save a .PKT file. NOTE: Peak list headings are not included when you save a .PKT file. If you require headings, copy the peak list directly to Excel instead of saving it as a .
Chapter 3 Pe ak Detecti on and Labeling 3- 42 Applied Biosystems 3 3.3.4 Sorting, Filtering, and Printing the Peak List Sorting the pea k list The peak l ist is dis played in order by i ndex num ber . Y o u can sort the list by any field by c licking th e column head er buttons (Fi gure 3-1 6).
Peak List Data Explorer ™ So ftw are User ’ s Guide 3-4 3 3 2. S elect Enable Peak List Filter , th en select: Filter T ype Descriptio n Monoi sotopic Labels the peak of the lowest detected mass in an isotope envelope.
Chapter 3 Pe ak Detecti on and Labeling 3- 44 Applied Biosystems 3 Printing the pea k list T o pr int the pe ak list: 1. Display the peak list as you want it printed.
Deisotoping a Spectrum Data Explorer ™ So ftw are User ’ s Guide 3-4 5 3 3.4 Deisotopi ng a S pectrum This sect ion incl udes: • Description • During pe ak deisotop ing • When to us e • Re.
Chapter 3 Pe ak Detecti on and Labeling 3- 46 Applied Biosystems 3 If the e xpected hig her theore tical peak masses and areas are present in the pea k list: • The pea k in questi on is co nsidered to be a monoi sotopic peak.
Deisotoping a Spectrum Data Explorer ™ So ftw are User ’ s Guide 3-4 7 3 Figure 3-17 Interpreting a Deis otop ed Trace Requirements The Deis otope func tion require s a singly char ged spectrum .
Chapter 3 Pe ak Detecti on and Labeling 3- 48 Applied Biosystems 3 Using the Deisotope function T o us e the Deis otope functi on: 1. Display the spectrum trace of interest. 2. Ma ke sure peak dete ction thres holds are set low en ough to detect the monoi sotopic peak befo re deisoto ping.
Deisotoping a Spectrum Data Explorer ™ So ftw are User ’ s Guide 3-4 9 3 CAUTION If you enter an invalid value in the Adduct field, for example, numbers, the spectrum is still converted to a deisotoped spectrum, and the peak height is proportional to the original peak area.
Chapter 3 Pe ak Detecti on and Labeling 3- 50 Applied Biosystems 3 Example F ig ur e 3- 19 an d Fig ure 3-2 0 illustrate the ef fects of deisotoping. B e fo re dei sot op ing (F ig ur e 3- 19), the s pectrum includes an isotope pattern with four detected peaks.
Deisotoping a Spectrum Data Explorer ™ So ftw are User ’ s Guide 3-5 1 3 Retu rning to th e origin al spect rum T o re turn to the original s pectrum: • If the orig inal sp ectrum wa s an unpr ocessed s pectrum, selec t Spectrum Numb er fro m the Disp lay menu.
Chapter 3 Pe ak Detecti on and Labeling 3- 52 Applied Biosystems 3 3.5 Peak Lab eling This se ction incl udes: • Charge s tate labels • Setting chro matogram and sp ec trum peak label s • Settin.
Peak Labeling Data Explorer ™ So ftw are User ’ s Guide 3-5 3 3 3.5.1 Charge St ate Labels Charge st ate labels A unique fe ature of th e Data Explorer s oftware is the ability to label m ultiply ch arged isot ope peaks with thei r charge state .
Chapter 3 Pe ak Detecti on and Labeling 3- 54 Applied Biosystems 3 • Isotopes must be r esolved • Peak dete ction parameters must be set to detect al l of the peaks i n the isot ope cluste r • Charge d etermina tion parameters m ust be se t appropri ately • Charge s tate labels must be enabl ed 3.
Peak Labeling Data Explorer ™ So ftw are User ’ s Guide 3-5 5 3 Figure 3-21 Chromatogram Peak Label Dialog Bo x 3. S elect Enable Labeling . 4. S et the numb er of decimal point s to be disp la ye d. 5. S elect Label Attribu tes: • Overlapping — Allows labels to be displ ayed when peaks are close together.
Chapter 3 Pe ak Detecti on and Labeling 3- 56 Applied Biosystems 3 8. Cli ck OK . The trace is displayed. The detected peaks t hat meet the peak labeling criteria are labeled. Setting spectrum labels T o la bel spectr um peaks: 1. Click the Spectrum window to activate it.
Peak Labeling Data Explorer ™ So ftw are User ’ s Guide 3-5 7 3 5. S elect the Mass T ype (peak apex or peak c entroid). 6. S elect the Peak Mass Label T ype: Label Descri ption Mass Labels with Apex or Centroid mass. NOTE: If you create a custom user label for a mass, the user label is displayed instead of the mass.
Chapter 3 Pe ak Detecti on and Labeling 3- 58 Applied Biosystems 3 7. Se lect lab el attrib utes: • Overl apping — Allows labels t o be displayed when peaks are close together. • Peak bounds — Displays peak start, peak end, and baseline. • Orie nta tion — Specifies Horizontal, 45-degree, or V e rtical label s.
Peak Labeling Data Explorer ™ So ftw are User ’ s Guide 3-5 9 3 If peak list filtering is enabled, only t he detected peaks that meet the peak filtering criteria are labeled according to the peak label settings. Otherwise, all detected peaks are labeled according to the peak label settings.
Chapter 3 Pe ak Detecti on and Labeling 3- 60 Applied Biosystems 3 Charge st ate not displayed If no char ge is dis played, th ere are a few possib le ca uses: • Peaks ar e more than 1 Da apart. • Filter wi dth is set too hi gh to detect o ther isotop e peaks.
Peak Labeling Data Explorer ™ So ftw are User ’ s Guide 3-6 1 3 3.5.3 Setting Custom Peak Labels This section incl udes: • Description • Customiz ing colo rs, font, and size • Creating cus tom peak labels • Applyin g user labels from .
Chapter 3 Pe ak Detecti on and Labeling 3- 62 Applied Biosystems 3 Figure 3-23 User Label Setup Dialog Bo x 4. Se lect t he Label T y p e (spectr a only) : • Mass — Labels with Apex or Centroid mass.
Peak Labeling Data Explorer ™ So ftw are User ’ s Guide 3-6 3 3 6. T o manu ally ent er label s ettings, clic k . The User Label Entry dialog box is displayed (Figure 3-24) . Figure 3-24 User Lab el Entry Dialog Box (Spectrum) 7. S et the foll owi ng parameter s: Spectrum Parameter Description/Specif ies Label T ext of the label to display .
Chapter 3 Pe ak Detecti on and Labeling 3- 64 Applied Biosystems 3 T o enter Peak Mass, X V alue, or Mass Difference, you can type values or right-click-drag over a pea k . A Mass T olerance or X T olerance of 1 is automatically entered when you right-click-drag.
Peak Labeling Data Explorer ™ So ftw are User ’ s Guide 3-6 5 3 Applying user labels from .LBC or .LBS files Y o u can apply labels yo u previo usly cre ated and s aved in .LBC or .LBS fil es. Open th e .LBC or .LBS file by c licking in the Use r Label Setup di alog box .
Chapter 3 Pe ak Detecti on and Labeling 3- 66 Applied Biosystems 3 User labels not displaye d If a user la bel is no t displaye d or is displ ayed inc orrectly , possib le cause s are: • User label s are not e nabled. • The pea k does n ot fall wit hin the sp ecified T oleran ce.
Process that Occurs D uring Peak Detection, Cent roidi ng, and I ntegratio n Data Explorer ™ So ftw are User ’ s Guide 3-6 7 3 3.6 Process that Occurs During Peak Detection, Centroid ing, and Integratio n This s ectio n gives an overvi ew of th e proc es s that oc curs durin g peak detec tion, centro iding and i ntegration.
Chapter 3 Pe ak Detecti on and Labeling 3- 68 Applied Biosystems 3 • For spec tral data, determi nes the pe ak boundar ies by one of two means: • If the Noise Threshold is greater than z ero, the software scans from the valle y regions toward the apex region using the number of dat a points defined by the Filter Widt h.
Process that Occurs D uring Peak Detection, Cent roidi ng, and I ntegratio n Data Explorer ™ So ftw are User ’ s Guide 3-6 9 3 After p eak detecti on A fter peaks are detecte d: • Centroid m ass is c alculated fo r spectral da ta, then modified b y Gaussi an peak fitti ng, if it i s selected .
Chapter 3 Pe ak Detecti on and Labeling 3- 70 Applied Biosystems 3 Int egra tion The Data Explorer software integrates ch romatogr aphic and spectral peaks to calcul ate the peak ar ea usin g one of t.
Default Pea k De tection S ettin gs Data Explorer ™ So ftw are User ’ s Guide 3-7 1 3 3.7 Default Peak Detectio n Settings This sect ion incl udes: • Default .SE T files prov ided • Additiona l V o yager .S ET files provided Default .SE T files pro vide d Defau lt peak de tection sett ings are c ontained in th e following .
Chapter 3 Pe ak Detecti on and Labeling 3- 72 Applied Biosystems 3 The foll owing table lis ts the default settings in the .SET files provid ed for sp ectra. Parameter Mariner Spectrum MARINER.S ET V oyager Spectr um Linear VOY AG ER LINEA R.SET Reflecto r VOY AGER REFLECTOR .
Default Pea k De tection S ettin gs Data Explorer ™ So ftw are User ’ s Guide 3-7 3 3 Additional Vo y a g e r . S E T f i l e s pro vide d Additi onal .SET file s that have been dev eloped for detec tion of different types of data are inc luded in the C:VOY AGERSE TTINGS d irectory .
Chapter 3 Pe ak Detecti on and Labeling 3- 74 Applied Biosystems 3.
4 Chapter Data Explorer ™ So ftw are User ’ s Guide 4 -1 4 Examining Chromatogram Dat a This chapter contains the following sections: 4.1 Overview ...................... ................. .................. 4-2 4.2 Creating an Extracted Ion Chromatogram .
Chapter 4 Exami ning Chr omatogr am Data 4- 2 Applied Biosystems 4 4.1 Ov erview This se ction incl udes: • T y pes of Ma riner data • T y pes of V oyag er data • Creatin g macros to combine pro.
Overview Data Explorer ™ So ftw are User ’ s Guide 4 -3 4 Y o u can display extracted chromatogr ams from Ma riner data file s by selecti ng the P rocess menu with a Chromato gram window d isplaye d, then selec ting: T ypes of V oyager data V oyager ch romato grams c an opti onall y be displ ayed f or multis pectrum .
Chapter 4 Exami ning Chr omatogr am Data 4- 4 Applied Biosystems 4 Y ou can disp lay the fo llowing t ypes of V oyag er data by selec ting Extracted I on from the P rocess menu with a Chromato gram wi.
Creating an Extract ed Ion Chro matogram Data Explorer ™ So ftw are User ’ s Guide 4 -5 4 4.2 Creating an Extracted Ion Chromatogram This sect ion incl udes: • Creating an E xtracted Ion Ch roma.
Chapter 4 Exami ning Chr omatogr am Data 4- 6 Applied Biosystems 4 4. In th e Extracted Ion Chromatogr am dialo g box (Figure 4-1 on page 4-7), select one of the follow ing from the Mass Range/D ifference T ype dro p-down lis t: • Center/Win dow , then type the mass of interest and the mass window for masses to include.
Creating an Extract ed Ion Chro matogram Data Explorer ™ So ftw are User ’ s Guide 4 -7 4 Figure 4-1 Extracted Ion Chromatogram Dialog Box 5. S pe ci fy the Ex trac tio n Mode: • Accumulative .
Chapter 4 Exami ning Chr omatogr am Data 4- 8 Applied Biosystems 4 Fro m the Spectrum window T o c reate an extra cted ion chr omatogram f or a mass ran ge from the Spectru m window: 1. Click the Chromatogram window to ac tivate it. 2. Se lect Duplicate Act ive T race fro m the Display m enu to keep the original da ta displaye d after proc essing.
Creating an Extract ed Ion Chro matogram Data Explorer ™ So ftw are User ’ s Guide 4 -9 4 4.2.2 Creating a Constant Neutral L oss (CNL) Chromatogram This sect ion incl udes: • Over view • Appl.
Chapter 4 Exami ning Chr omatogr am Data 4- 10 Applied Biosystems 4 Labeling spectrum peaks with mass difference (optional) Y ou can label spectrum peaks with m ass difference s to ass ist you in determin ing the ma ss differe nce s to spec if y in the CNL extracted ch ro mato gr am.
Creating an Extract ed Ion Chro matogram Data Explorer ™ So ftw are User ’ s Guide 4-1 1 4 5. T y pe in the Mass Difference and T olerance. NOTE: Do not right-click-drag across a peak in the Spectrum window to select the mass difference. Figure 4-3 Extracted Ion Chromatogram Dialog Box with Neutral Loss Selected 6.
Chapter 4 Exami ning Chr omatogr am Data 4- 12 Applied Biosystems 4 Example Figure 4-4 shows a TIC that co ntains three fla vonoid compound peaks. T o determine if the digly cosyl gr oup has fragment ed from the paren t ion in any of these com pounds, you can generate a CNL extracted chromatogr am.
Creating a n Extract ed A bsorb ance Chro matogr am ( XAC ) (Mariner Dat a Only) Data Explorer ™ So ftw are User ’ s Guide 4-1 3 4 4.3 Creating an Extracted Absorb ance Chrom atogram (XAC) (Marine.
Chapter 4 Exami ning Chr omatogr am Data 4- 14 Applied Biosystems 4 Figure 4-6 Extracted Ab sorbance Chromatogram Dialog Box 6. Sp ecify the E xtraction Mode: • Accumulative — Creat es a single trace combining intensities of all specified wavelengths • Indi vidual — Crea tes one trace for eac h specified wavelength 7.
Creating a n Extract ed A bsorb ance Chro matogr am ( XAC ) (Mariner Dat a Only) Data Explorer ™ So ftw are User ’ s Guide 4-1 5 4 From the Spectrum window T o c reate an extracted abs orbance chromatogram for a wavele ngth range from the Spe ctrum windo w: 1.
Chapter 4 Exami ning Chr omatogr am Data 4- 16 Applied Biosystems 4 Figure 4-7 Extracted Ab sorbance Chromatogram 7. T o retur n to the o riginal trace, s ee “ Returni ng to the origin al trace ” on p age 4-4.
Noise Filteri ng/S moothin g Data Explorer ™ So ftw are User ’ s Guide 4-1 7 4 4.4 Nois e Filtering/ Smoothing Descr iption The Noi se Filter/Smo oth comman d provides th ree optio ns for reduc in.
Chapter 4 Exami ning Chr omatogr am Data 4- 18 Applied Biosystems 4 2. Se lect the m ethod to use based upo n the type o f data you are exa mining, th en enter t he associa ted value display ed for the m ethod you sel ect: T ype of Data Suggested Method Description Higher resolution data Noise Filter (NF) (May affect peak resolution.
Noise Filteri ng/S moothin g Data Explorer ™ So ftw are User ’ s Guide 4-1 9 4 3. Click OK . The trace is displayed with an NF, SM, or NR tr ace label. 4. T o re turn to the origina l trace, see “ Retur ning to the original trace ” on page 4- 4.
Chapter 4 Exami ning Chr omatogr am Data 4- 20 Applied Biosystems 4 4.5 Adding and Su btracting Raw Spe ctra With in a Data File Use the A dd/Subtract Spectra c ommand to m anipulate r aw spectra wi thin a singl e data file.
Adding a nd Subtr acting Raw Spectra Withi n a Dat a Fil e Data Explorer ™ So ftw are User ’ s Guide 4-2 1 4 Figure 4-9 . Add and Subtract Spectra Dial og Box 4. S elect spec tra to add by doing on e of the followi ng: • Right-click-drag the area of the trace in the Chromatogram window .
Chapter 4 Exami ning Chr omatogr am Data 4- 22 Applied Biosystems 4 NOTE: Before you can subtract spectra, you must firs t specify spectra to be added. 6. Se lect spe ctra to sub tract as descr ibed in step 4. 7. Se lect the A dd/Su btract mode: • A verage — Spectra in each list are averaged before the addition or subtraction occurs.
Displaying MS Method Data (Mar iner Data Only) Data Explorer ™ So ftw are User ’ s Guide 4-2 3 4 4.6 Displaying MS Metho d Data (Mariner D a ta Only) Overview If you acq uired a data f ile using an MS Metho d and ass igned event tags, you can display c hromatogra m traces in Data Explo rer filtered by event tag.
Chapter 4 Exami ning Chr omatogr am Data 4- 24 Applied Biosystems 4 Hint: Add mode is useful when you filter the same trace for different event t ags. The original trace remains displ ayed and accessible. Each filtered trace (up to four total t races) is added, allowing for visual comparison.
Displaying MS Method Data (Mar iner Data Only) Data Explorer ™ So ftw are User ’ s Guide 4-2 5 4 NOTE: Only tags present in the data file are available. 6. Sel ect the ev ent tags to disp lay , then c lick OK . The filtered trace is displayed with an EF trace label (Figure 4-1 1).
Chapter 4 Exami ning Chr omatogr am Data 4- 26 Applied Biosystems 4 Hint: Add mode is useful when you are filtering the same trace for different event t ags. The original trace remains displayed and accessible. Each filtered trace (up to four tot al traces) is added, allowing for visual comparison.
Adjusting the B aselin e Data Explorer ™ So ftw are User ’ s Guide 4-2 7 4 4.7 Adjusting the Baselin e This sect ion incl udes: • Usin g Base line Of fset • Using Baselin e Correc tion 4.
Chapter 4 Exami ning Chr omatogr am Data 4- 28 Applied Biosystems 4 Figure 4-12 Baseli ne Offset Dialog Bo x 4. Rig ht-click- drag the left baselin e to offset. The selecte d value i s displayed i n the Left B aseline fi eld. 5. Rig ht-click- drag the ri ght baseline to offset.
Adjusting the B aselin e Data Explorer ™ So ftw are User ’ s Guide 4-2 9 4 4.7.2 Using Baseline Correction Descr iption The B aseline Cor rection fea ture is a fu nction tha t corrects for a cu rved base line, inc luding a DC-offset base line, by elimi nating broad artifacts from the data set.
Chapter 4 Exami ning Chr omatogr am Data 4- 30 Applied Biosystems 4 4.8 Using UV Trac e Offset (Mariner Data Only) T o al ign a UV t race with a c hromato gram trace: 1.
Using UV Trace Of fset (Mariner Data Only) Data Explorer ™ So ftw are User ’ s Guide 4-3 1 4 6. Click OK . The UV trace peak is shifted to align with the ch ro ma tog ra m trace peak. NOTE: T o restore the original UV tr ac e, open the UV T race Offset dialog box (see step 4), then click Reset .
Chapter 4 Exami ning Chr omatogr am Data 4- 32 Applied Biosystems 4.
5 Chapter Data Explorer ™ So ftw are User ’ s Guide 5 -1 5 Examining Spectrum Dat a This chapter contains the following sections: 5.1 Overview ...................... ................. .................. 5-2 5.2 Creating a Combined Spectrum .......
Chapter 5 Exa mining Spectr um D ata 5- 2 Applied Biosystems 5 5.1 Ov erview T ypes of spectra you can display Y ou can displa y the fo llowing type s of spectr um data: • Single spectrum — Double-click any point in th e TIC to displ ay the co rresponding spectru m.
Overview Data Explorer ™ So ftw are User ’ s Guide 5 -3 5 Retu rning to th e origin al spect rum M any process ing funct ions gene rate a new tr ace. If you ha ve T rac e Replace mo de set to Repl ace, the ne w trace replaces the ori ginal trace. F or informati on on Repl ace mode, se e Secti on 2.
Chapter 5 Exa mining Spectr um D ata 5- 4 Applied Biosystems 5 5.2 Creatin g a Combined Spectrum NOTE: Befor e creating a combined spectrum for V oyager multispectrum data files, calibrate th e data. See Section 5.3, Manual Calibration. T o c reate a comb ined spe ctrum: 1.
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5 -5 5 5.3 Manu al Calibration This sect ion describe s: • Overvie w of manual calibrati on • Manually calibrati ng • Creating o.
Chapter 5 Exa mining Spectr um D ata 5- 6 Applied Biosystems 5 The man ual calibr ation featu re provid es two modes for peak match ing: • Automatic — The software au tomatica lly com pares refere nce masses to observ ed masse s, and lists p eaks that are within the spec ified peak m atching c riteria.
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5 -7 5 5.3.2 Manually Calibrating This sect ion descr ibes: • Before c alibrating V oy ager data • Manually calibrati ng a single spectrum • Appl ying n ew const ant s to th e data file • Exporti ng calib r atio n con stants (.
Chapter 5 Exa mining Spectr um D ata 5- 8 Applied Biosystems 5 Manually calibrating a single sp ectrum NOTE: Multi- point calibration yields higher mass accuracy than one-point calibration. Selecting calibrant peaks that bracket the mass of interest also yields higher mass accuracy .
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5 -9 5 Figure 5-2 Manual Mass C alibration Dialog Box 4. S elect a cal ibratio n referenc e file. For in formation on creat ing a refere nce file, s ee Section 5.3.3, Creating or Modify ing a Cali bration Reference File (.
Chapter 5 Exa mining Spectr um D ata 5- 10 Applied Biosystems 5 6. Se lect t he Peak We i ghting Fa ct or . If the calibrati on include s more than two po ints, you c an apply the followin g weighting.
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-1 1 5 8. D o any of the followin g: • Click OK to accept the selected reference mass for matching, then add it t o the Peaks Matched list. • Select a differ ent reference mass, then click OK .
Chapter 5 Exa mining Spectr um D ata 5- 12 Applied Biosystems 5 9. Rep eat step 7 and step 8 until al l desired peaks are in the matched l ist. Eliminat ing data points 10.
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-1 3 5 Figure 5-4 Eliminate Fit Outlier D eletes the Match with the L argest Fit Error from the Ou tput Window T o clear the entire list, click Delete Entire Lis t . Plotti ng 1 1. T o apply the calibration constants to the displayed spectrum, click Plot .
Chapter 5 Exa mining Spectr um D ata 5- 14 Applied Biosystems 5 Figure 5-5 Cali bration S tatistics in Output Wi ndow If you calibrate more than once, subsequent calibration statistics are added to the end of the list in the Output window. Older calibration stat i stics are listed at the top of the list.
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-1 5 5 NOTE: If you are calibrating Mariner data, see “ Ensuring that masses match during calibration ” on p age 5-24.
Chapter 5 Exa mining Spectr um D ata 5- 16 Applied Biosystems 5 Exporting calibration constants (.CAL file) The cal culated calibrati on constants can be exporte d to a .
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-1 7 5 5. T o s ave the cal ibration to the data file, select Mass Calibration from the Proce ss menu, the n: 5.3.3 Creating or Modifying a Calibration Reference F ile (.REF) This sect ion incl udes: • Definition • .
Chapter 5 Exa mining Spectr um D ata 5- 18 Applied Biosystems 5 Definition A calibrati on referenc e file (. REF) is a li st of mass es and corresp onding in formation from which you can sel ect referen ce mass es during calibr ation.
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-1 9 5 The Edit/Create Reference Peak Information dialog box (Figure 5-6) is displayed. Figure 5-6 Edit/Create Referen ce Peak Information Dialog Bo x 2. Ty p e the Name and Th eoretical m/z for a re ference compou nd, then se lect the c harge state .
Chapter 5 Exa mining Spectr um D ata 5- 20 Applied Biosystems 5 CAUTION The software allows you to add multiple items with t he same m/z value to the reference file if any other attribute of the refer ence compound is different (for example, charge state or name).
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-2 1 5 3. T o m odify an entr y , click th e entry to sele ct it, mod ify the entry as nee ded, then c lick Up date . 4. T o d elete an e ntry , click the entry to selec t it, then cl ick Del ete .
Chapter 5 Exa mining Spectr um D ata 5- 22 Applied Biosystems 5 5.3.4 Reverting to Instrument Calibration The Rever t to Instrume nt Calibrati on functio n does the followin g: • Marine r data — Reapplies the orig inal calib ration constants use d to acquir e the data.
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-2 3 5 If you apply the calibration, the next time you open the data file, the MC tr ace label is not displayed. V oyager data Apply Calibration The current spectrum is calibr ated and displayed with an MC trace label.
Chapter 5 Exa mining Spectr um D ata 5- 24 Applied Biosystems 5 5.3.5 Hint s for Calibrating Mariner Dat a Ensuring that mass es matc h during calibration Mariner TOF Analyz er p arameters affect flight tim es of ions.
Manual Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-2 5 5 5.3.6 Hint s for Calibrating V o yager Dat a Importing a calibrati on I f you impo rt a calibra tion, you must import a calibr.
Chapter 5 Exa mining Spectr um D ata 5- 26 Applied Biosystems 5 5.4 Automatic Calibration This se ction incl udes: • Overvi ew of automa tic calibr ation • Importi ng and speci fying automa tic calibrat ion setti ngs • Auto matical ly cali brati ng (Marin er dat a only ) NOTE: Automatic c alibration is not supported for Mariner DAD dat a.
Automatic Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-2 7 5 • Matches all peaks that meet the specified Reference Matching criteria.
Chapter 5 Exa mining Spectr um D ata 5- 28 Applied Biosystems 5 When to use Use au tomatic cal ibration fo r Mariner data when you: • Have many spec tra to calibrat e • Know in advanc e what refer.
Automatic Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-2 9 5 5.4.2 Importing and Specifying Automatic Calibration Settings Importing Hint: Importing automatic calibration settings is useful when you calibrate batches of related s amples.
Chapter 5 Exa mining Spectr um D ata 5- 30 Applied Biosystems 5 2. F rom the Peak s menu, sel ect Peak Label , then set t he Mass La bel T y pe to Centroi d . NOTE: For spectr a containing broad peaks that have unresolved adducts or impurities such as proteins, you may obtain better result s if you use apex instead of centroid settings.
Automatic Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-3 1 5 5. T o add up to 10 referenc e masses to the Mass es to Match list, do e ither of th e following: • Click Add All to add the first 10 reference masses from the reference file.
Chapter 5 Exa mining Spectr um D ata 5- 32 Applied Biosystems 5 6. Sp ecify refe rence mass es to add by doing ei ther of the followin g: • Cli ck a ma ss, the n clic k OK . • T ype new reference mass information in the Name, Theoretical m/z, Charge, and Elemental Composition fields, then click OK .
Automatic Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-3 3 5 10. S elect the Peak W eighting Factor . If the calibration includes more than two points, you can apply the following weig.
Chapter 5 Exa mining Spectr um D ata 5- 34 Applied Biosystems 5 5.4.3 Automatically Calibrating (Mariner Dat a Only) This se ction incl udes: • Autom atically c alibratin g • Applyi ng new co nsta.
Automatic Calibr ation Data Explorer ™ So ftw are User ’ s Guide 5-3 5 5 Applying new constants to the data file T o save the calibr ation co nstants for each spectru m in the d ata file, se lect Apply Calibration from th e Process m enu. Calibrati on results Automat ic calibr ation resu lts are displ ayed in the O utput window (Fig ure 5-9 ).
Chapter 5 Exa mining Spectr um D ata 5- 36 Applied Biosystems 5 5.5 Centroid ing NOTE: Centroiding is not supported for Mariner DAD dat a. T o displa y peaks as centroid traces: 1. Click the Spectrum window to activate it. 2. Se lect Duplicate Act ive T race fro m the Display m enu to keep the original da ta displaye d after proc essing.
Mass Deconvolution (Mariner D at a Only) Data Explorer ™ So ftw are User ’ s Guide 5-3 7 5 5.6 Ma ss Deconv olution (Marin er Dat a Only) NOTE: Mass deconvolution is not supported for Mariner DAD data. NOTE: The Mass Deconvolution software is an option in the Data Explorer software.
Chapter 5 Exa mining Spectr um D ata 5- 38 Applied Biosystems 5 The Multiply Charged Deconvolution dialog box (Figure 5-1 1) is display ed. 4. In t he Spectrum window , right- click- drag one mul tiply charged peak.
Mass Deconvolution (Mariner D at a Only) Data Explorer ™ So ftw are User ’ s Guide 5-3 9 5 6. S elect the me thod to use for calculat ion: • Automat ic — Selects additional multiply charged peaks based on the selected peaks and performs the calculation.
Chapter 5 Exa mining Spectr um D ata 5- 40 Applied Biosystems 5 1 1. C lick OK . The result is displayed in the Output window and the zero-charge spectrum is displayed with a DECONV trace label, if selected. NOTE: T he numerical result displayed in the output window generally is more accurate than the computer-generated spectrum.
Mass Deconvolution (Mariner D at a Only) Data Explorer ™ So ftw are User ’ s Guide 5-4 1 5 5. Ty p e values for th e followi ng masses f or the gener ated zero-c harge spectr um: • Center — Ce.
Chapter 5 Exa mining Spectr um D ata 5- 42 Applied Biosystems 5 5.7 Noise Filt ering/Smo othing Description The Noise Filt er/Smoot h function include s four op tions for reduci ng noise in a spectrum.
Noise Filteri ng/S moothin g Data Explorer ™ So ftw are User ’ s Guide 5-4 3 5 3. S elect the me thod to use based on the type o f data you are ex amining, then enter the associ ated value d ispla.
Chapter 5 Exa mining Spectr um D ata 5- 44 Applied Biosystems 5 4. Cli ck OK . The trace is displayed with an RSM, NF, NR or SM trace label. 5. T o retur n to the o riginal trace, s ee “ Returni ng to the origin al spect rum ” on page 5-3. High-resolution data Noise Removal (NR) (Does not affect peak resolution.
Adjusting the B aselin e Data Explorer ™ So ftw are User ’ s Guide 5-4 5 5 5.8 Adjusting the Baselin e This sect ion incl udes: • Usin g Base line Of fset • Using Baselin e Correc tion • Using Adv anced B aseline Cor rection 5.
Chapter 5 Exa mining Spectr um D ata 5- 46 Applied Biosystems 5 Figure 5-14 Baseli ne Offset Dialog Bo x 4. Rig ht-click- drag the left baselin e to offset. The selecte d value i s displayed i n the Left B aseline fi eld. 5. Rig ht-click- drag the ri ght baseline to offset.
Adjusting the B aselin e Data Explorer ™ So ftw are User ’ s Guide 5-4 7 5 5.8.2 Using Baseline Correction Descr iption The B aseline Cor rection function co rrects for a cu rved baseli ne, incl uding a DC-offset baseline, by elimin ating broad artifa cts from the data set.
Chapter 5 Exa mining Spectr um D ata 5- 48 Applied Biosystems 5 5.8.3 Using Advanced Baseline Correction This se ction incl udes: • Description • When to u se • Correcti ng the bas eline • Gen.
Adjusting the B aselin e Data Explorer ™ So ftw are User ’ s Guide 5-4 9 5 When to use Use advanced ba seline c orrectio n if you a re analyzi ng data with an o f fs et in the spectrum, partic ularly da ta with a strong sloping b aseline at low mass.
Chapter 5 Exa mining Spectr um D ata 5- 50 Applied Biosystems 5 3. En ter parameters a s described below . These parameters interac t with each ot her and req uire exp erimentatio n to determi ne the optimum settings for your data. Refe r to “ General gu idelines for settin g parameters ” on page 5-54 for more in formation.
Adjusting the B aselin e Data Explorer ™ So ftw are User ’ s Guide 5-5 1 5 Peak Width (at half h eight) (continued) Set Peak Width according to the data you are correcting: • For best results, set to the peak width at half height of the narrowest peak.
Chapter 5 Exa mining Spectr um D ata 5- 52 Applied Biosystems 5 Flexibility With the Peak Width p arameter , determines the number of point s us ed to estimate the baseline amplitude at regularly spaced point s in the spectrum, but is not directly proportional to the number of points used.
Adjusting the B aselin e Data Explorer ™ So ftw are User ’ s Guide 5-5 3 5 NOTE: Flexibility and Degree parameters may require some testing to determine the optimum settings for your data. 4. Click OK . The ba seline is adjusted, and the trace is d isplayed w ith a AdvBC trace label.
Chapter 5 Exa mining Spectr um D ata 5- 54 Applied Biosystems 5 General guidelines for setting parameters Refer to th e following ta ble to dete rmine how to set Adva nce Base line Corre ction parameter s and ob tain the desi red baselin e correc tion.
Adjusting the B aselin e Data Explorer ™ So ftw are User ’ s Guide 5-5 5 5 Troubleshooting If the baseline r ises preced ing and fol lowing a peak a fter the correcti on (a “ hump ” u nder the.
Chapter 5 Exa mining Spectr um D ata 5- 56 Applied Biosystems 5 5.9 Truncatin g a Spectrum Description The T runca te functio n remove s data points from a trace outside a se lecte d region.
Truncating a Spectrum Data Explorer ™ So ftw are User ’ s Guide 5-5 7 5 The data in the spectrum is truncated t o the selec ted range and is dis pla ye d w ith a T R tr ac e l abel. T h e r ange displayed on the axis of the truncat ed trace is the range of the original data f i le, and may be wider than the range of the truncated spectrum.
Chapter 5 Exa mining Spectr um D ata 5- 58 Applied Biosystems 5 Figure 5-19 Tru ncated S pectrum — Low Mass Gate Spike Elimi nated.
Converting to a Singly Charged Spectrum (Mariner Data Only) Data Explorer ™ So ftw are User ’ s Guide 5-5 9 5 5.10 Convertin g to a Sin gly Charge d Spectrum (Mariner Data Only) NOTE: Single-charge conversion is not supported for Mariner DAD dat a.
Chapter 5 Exa mining Spectr um D ata 5- 60 Applied Biosystems 5 3. Se lect Duplicate Act ive T race fro m the Display m enu to keep the original da ta displaye d after proc essing. 4. F rom the Pr ocess menu , select Single-Charge Conversion . The Single-Charge Spectrum Conversion dialog box (Figure 5-20) is displayed.
Converting to a Singly Charged Spectrum (Mariner Data Only) Data Explorer ™ So ftw are User ’ s Guide 5-6 1 5 Example F igu re 5 -2 1 and F ig ur e 5- 22 illustrate the effects of single-charge conversion. Be fo re c on ve rs io n (F ig ur e 5-2 1) , the spectrum includes +2 and +3 charged species of neurotensin.
Chapter 5 Exa mining Spectr um D ata 5- 62 Applied Biosystems 5 NOTE: Charge states other than 0 or 1 in the converted trace indicate that a peak in the original spectrum is labeled with an incorrect charge state. Set peak detection thresholds to disregard these peaks and convert the spectrum again.
AutoSatur ation Corre ction (Mariner Data Only) Data Explorer ™ So ftw are User ’ s Guide 5-6 3 5 Ef fect on Mariner .RST files The AutoS aturatio n Correction f eature is no t applied to .RST file s save d from the Mar iner Instr ument Cont rol Pane l, even when Sat uration C orrection is turned o n.
Chapter 5 Exa mining Spectr um D ata 5- 64 Applied Biosystems 5 5.12 Adding and Subtrac ting Raw or Proc essed Spect ra from the Same o r Differ ent Dat a Files (Dua l Spectral Tr ace Arithmet ic) The Dual Spectral T r ace Arith metic fun ction le ts you add two spectra to gether , or su btract one s pectrum from another .
Adding and S ubtractin g R aw or P ro cessed Spectr a fr om th e Same or Differ ent Data F iles ( Dual Data Explorer ™ So ftw are User ’ s Guide 5-6 5 5 Figure 5-23 Dual Spectral T race Arithmetic Dialog Box 6. S et the Mass T olerance with in which d ata points from the different trac es will be c onsidere d as the same mass.
Chapter 5 Exa mining Spectr um D ata 5- 66 Applied Biosystems 5.
6 Chapter Data Explorer ™ So ftw are User ’ s Guide 6 -1 6 Using T ools and Applications This chapter contains the following sections: 6.1 Usi ng the Elemental Composition C alculator .......... 6-2 6.2 Usi ng the Isotope Calculator ..............
Chapter 6 Using T ools an d A pplicati ons 6- 2 Applied Biosystems 6 6.1 U sing th e Elemen tal Composit ion Calculator This se ction incl udes: • Determ i ni ng ele men tal co mpo sit io n • Sett ing limits 6.
Using the Elemental Composition C alcul ator Data Explorer ™ So ftw are User ’ s Guide 6 -3 6 Procedure T o d etermine elemental comp osition: 1. Dis play the sp ectrum con taining the pea k of inter est. 2. C lick the Spectrum w indow to ac tivate it.
Chapter 6 Using T ools an d A pplicati ons 6- 4 Applied Biosystems 6 4. En ter m/z v alues in th e m/z ra tio list by doing any of the followin g: • Right-click-drag over a peak in the spectrum to add the m/z and the associated charge st ate.
Using the Elemental Composition C alcul ator Data Explorer ™ So ftw are User ’ s Guide 6 -5 6 •“ A dding new elements and setting limits ” on page 6-9 •“ S etting limits for other result types ” on page 6-12 1 1. Click More P arameters , then enter the Minimum and Maximum Double Bond Equivalents to include in the calculation.
Chapter 6 Using T ools an d A pplicati ons 6- 6 Applied Biosystems 6 Hint: Y ou can sort the results in a column by clicking the column header . Result s in clude: • Index — Seque ntial num ber assig ned to each result. • Input m/z — Entere d m/z for ea ch compo sition calculati on.
Using the Elemental Composition C alcul ator Data Explorer ™ So ftw are User ’ s Guide 6 -7 6 Displaying t he theoretical isotope distribution T o d isplay the theoreti cal isotope distribution for a calc ulated formul a, double- click th e correspond ing line in the Eleme ntal Analys is ta b of th e Output window .
Chapter 6 Using T ools an d A pplicati ons 6- 8 Applied Biosystems 6 2. T o c hange the l imits for an elem ent, double -click a n element to display the Isoto pe dialog bo x (Fig ure 6-4 ). Figure 6-4 Isoto pe Dialog Box NOTE: Ignore the column of check boxes to the left of the Isotope column if it is displayed.
Using the Elemental Composition C alcul ator Data Explorer ™ So ftw are User ’ s Guide 6 -9 6 Adding new elements and setting limits T o a dd new ele ments and set limits: 1. T o add new elements and set limits for Elemental results, click Element Limits in the E le men ta l Co mpo s iti on Ca l cul at or dialog box.
Chapter 6 Using T ools an d A pplicati ons 6- 10 Applied Biosystems 6 Figure 6-6 P eriodic Table 3. Cli ck an e lement to sel ect it and to displ ay the Isotop e dialog b ox (Fi gure 6- 7).
Using the Elemental Composition C alcul ator Data Explorer ™ So ftw are User ’ s Guide 6-1 1 6 Figure 6-7 Isotope Dialo g Box NOTE: Ignore the column of check boxes to the left of the Isotope column if it is displayed. 4. C hange the M inimum an d Maximu m number of occurr ences for t he first isoto pe of the el ement as n eeded.
Chapter 6 Using T ools an d A pplicati ons 6- 12 Applied Biosystems 6 Setting limits for other result types T o s et lim its for amin o acid, DNA , RNA, o r carboh ydrate r esult types: 1. Click the limits button displayed for the select ed result type in the Ele me nt al Co mp osi tio n C al cu la tor dialog box (see Figure 6-1 on page 6-3).
Using th e Isot ope Calculator Data Explorer ™ So ftw are User ’ s Guide 6-1 3 6 6.2 Using the Isotope Calculator Descr iption Use the Isotope c alculator t o generate a theoretic al isotope distribu tion. Y ou c an compare or overlay th e theoretic al distribu tion with you r observ ed distributio n.
Chapter 6 Using T ools an d A pplicati ons 6- 14 Applied Biosystems 6 4. Se lect t he Formula or Sequenc e for the type o f isotope to calcul ate. 5. Se lect a for mula from the list, or type in a new formula . V alid en tries for e ach formul a type are: 6.
Using th e Isot ope Calculator Data Explorer ™ So ftw are User ’ s Guide 6-1 5 6 7. S pecify the Add/Su btract Group option. T o en able or disabl e the opti on, select or d eselect the Group T y pe check b ox.
Chapter 6 Using T ools an d A pplicati ons 6- 16 Applied Biosystems 6 10. Select the calculation mode: • FWHM — Resolves peaks using the full peak width at peak half height. • 10% V alley — Resolves peaks to a 10 percent valley . 1 1. Set the Threshold %.
Using th e Isot ope Calculator Data Explorer ™ So ftw are User ’ s Guide 6-1 7 6 T o calculate a theoretical isotope distribution for doubly sodiated and deprotonated β -cyclodextrin with – 1 c.
Chapter 6 Using T ools an d A pplicati ons 6- 18 Applied Biosystems 6 Evalua ting trac es The theo retical is otope dist ribution is displayed in the Spectr um window with an ISO trac e label (Fig ure 6 -10). Figure 6-10 Isotope Tr ace If you hav e the Repl ace mode set to Add in the Displ ay T race dialog b ox, a new tr ace is add ed.
Using th e Isot ope Calculator Data Explorer ™ So ftw are User ’ s Guide 6-1 9 6 Results Th e results of the ca lculation are di splaye d in the Result tab of Output wind ow (Figure 6-1 1).
Chapter 6 Using T ools an d A pplicati ons 6- 20 Applied Biosystems 6 6.3 Using the Mass Reso lution Calculator Calculating mass resolution T o calcu late mass re solution : 1. Display the spectrum of interest. Make sure the Spectrum window is active.
Using the Mass R esolution Calculator Data Explorer ™ So ftw are User ’ s Guide 6-2 1 6 Figure 6-12 Mass Resolu tion Calculator 5. Spe cify the p eak for w hich to c alculate resolut ion by doing one of the followin g: • T ype in a Mass/Charge value.
Chapter 6 Using T ools an d A pplicati ons 6- 22 Applied Biosystems 6 The result is displayed in the Output window (Figure 6-13). Figure 6-13 Re solution Calculator Results Resolution result s calcul .
Using the S ignal-to- Noise Ra tio Calculator Data Explorer ™ So ftw are User ’ s Guide 6-2 3 6 6.4 Using the Signal-to-Noise Ratio Calcul ator Descr iption A sig nal-to-noi se ratio is typica lly used to descr ibe how wel l a peak of interest in a spectru m or chromatog ram is distin guished fro m backg round noise .
Chapter 6 Using T ools an d A pplicati ons 6- 24 Applied Biosystems 6 3. Se le ct the me th o d to u se , th en right-click-drag on peaks in t he tr ace t o enter the associat ed valu es disp layed for the met hod you sel ect: • Auto — Right-click-drag across the apex of the peak for signal-to-noise calculation.
Using the Ion Fragmentation Calculator Data Explorer ™ So ftw are User ’ s Guide 6-2 5 6 6.5 Using th e Ion Fragmentation Calculator Descr iption The Ion Fragmentat ion calcul ator gene rates a list o f possib le fragme nt masses fo r a peptide s equence you enter .
Chapter 6 Using T ools an d A pplicati ons 6- 26 Applied Biosystems 6 Figure 6-15 Ion F r agmentation Calc ulator Dialog Box 4. T ype or copy the amino ac id or res idue sequenc e of interes t. Use singl e-letter c odes. Sequence codes are case-sensitive.
Using the Ion Fragmentation Calculator Data Explorer ™ So ftw are User ’ s Guide 6-2 7 6 Setting Op tions 7. C lick Opt ions . The I on F rag me nt at io n Op tio ns dialog box (Figure 6-16) is displayed. Figure 6-16 Ion Fragmentat ion Options Dialog Box 8.
Chapter 6 Using T ools an d A pplicati ons 6- 28 Applied Biosystems 6 Setting user-d efine d amino acids 13. Click User-Defined Amino Acids . The User-Defined Amino Acids dialog box (Figure 6-17) is displayed. Figure 6-17 User-Def ined Amino Acids Dialog Box 14.
Using the Ion Fragmentation Calculator Data Explorer ™ So ftw are User ’ s Guide 6-2 9 6 Figure 6-18 Ion Fragmen tation Results for Synthetic Peptide (PPPPP PPPPPPP AR ) Results Resu lts are dis pl ayed in the: • Ions t able — L ists the masse s for each fragment a nd ion type.
Chapter 6 Using T ools an d A pplicati ons 6- 30 Applied Biosystems 6 Labeling peaks Click Label Peaks . The io n peaks specifi ed in the Opt ions dialog b ox are l abeled on the trace if they are pr esent (Figure 6-19) . Hint: T o screen out labels, decrease the Label T olerance in the Options dialog box.
Using the Ele men tal Targ eting Application Data Explorer ™ So ftw are User ’ s Guide 6-3 1 6 6.6 Using th e Elemental Targeting A pplication Descr iption The E lemental T argetin g applic ation determ ines if o bserved masse s in a spec trum corres pond to c hemical for mulas you enter .
Chapter 6 Using T ools an d A pplicati ons 6- 32 Applied Biosystems 6 Note the following when enter i ng formulas: • Spaces do not matter for formula.
Using the Ele men tal Targ eting Application Data Explorer ™ So ftw are User ’ s Guide 6-3 3 6 Displayi ng results T he res ults of the ca lculati on are disp layed in the E le m en ta l T a r ge t tab of t he Output w indow (Figur e 6-2 1).
Chapter 6 Using T ools an d A pplicati ons 6- 34 Applied Biosystems 6 6.7 Using the Macro Recorder Description The Macro Recor der featur e in Data Explorer allows yo u to set up mul ti-step tasks to execut e automat ically wh en you click a m acro bu tton.
Using the Macro Recorder Data Explorer ™ So ftw are User ’ s Guide 6-3 5 6 Location of macros All macr os you re cord are s tored in a file call ed DA T AEXPLORER .VB6 in the C:M ARINERPR OGRAM or C:VOY AGER directo ry. Displaying t he macro toolbar If the macro tool bar (Figure 6-22) is not display ed: 1.
Chapter 6 Using T ools an d A pplicati ons 6- 36 Applied Biosystems 6 Functions you perform in the Ou tput window , fo r example, sortin g or copying t he peak li st, are not supporte d by the Macro R ecorder . V i ew No commands supported Disp lay • Add/Remove Tr aces NOTE: Th e and butt ons are s upported by the Macr o Edito r .
Using the Macro Recorder Data Explorer ™ So ftw are User ’ s Guide 6-3 7 6 6.7.2 Recording a Macr o T o record a macro: 1. Open a dat a file. 2. F rom the T ool s menu, s elect Record New Macro . The Record Macro dialog box (Figure 6-23) is displayed.
Chapter 6 Using T ools an d A pplicati ons 6- 38 Applied Biosystems 6 6.7.3 Assigning Macros to Buttons Onl y macros presen t in th e DA T AEXPLOR ER.VB6 f ile can be assign ed to button s and run in the Data Expl orer software . NOTE: If you have installed a new version of Data Explorer software, new macros may be provided.
Using the Macro Recorder Data Explorer ™ So ftw are User ’ s Guide 6-3 9 6 De-assigning a macro from a button T o de-assign a macr o from a bu tton, sele ct the macro butt on in th e Assign Macro dialog box, then click De-assign .
Chapter 6 Using T ools an d A pplicati ons 6- 40 Applied Biosystems 6 Figure 6-25 Macros Dialog Bo x 3. Se lect t he macro to run from the list. 4. Cli ck Run . The macro executes. If t h e m a cr o contains a syntax error If the mac ro contains a s yntax error , it may c ause the Data Explorer software to close u nexpectedly .
Using the Macro Recorder Data Explorer ™ So ftw are User ’ s Guide 6-4 1 6 6.7.5 Deleting a Macro T o d elete a ma cro: 1. From the T ools menu, select Macros . The Macros dialog box (Figure 6-26) i s displayed. Figure 6-26 Macros Dialo g Box 2. S elect the ma cro to de lete from the list.
Chapter 6 Using T ools an d A pplicati ons 6- 42 Applied Biosystems 6 6.7.6 Advanced Macro Editing Access ing the V isual Basi c Editor Y ou can acces s the Visual Ba sic Edi tor to enhanc e or edit a script cre ated by th e Macro Rec order in Data Exp lorer , or to create a ne w script.
Using the Macro Recorder Data Explorer ™ So ftw are User ’ s Guide 6-4 3 6 6.7.7 Importing or Exporting Macros in DA T AEXPLORER.VB6 Y o u can impor t macros into, or expo rt macros fr om, the DA T AEXP LORER.VB6 project for us e in the Da ta Explorer software .
Chapter 6 Using T ools an d A pplicati ons 6- 44 Applied Biosystems 6 The selected macros are imported into the DA T AEXPLORER.VB 6 project. The .BAS files are included in the Modules folder in the DataExplorerProject, and the .FRM files are included in the Forms folder in the DataExplorerProject.
Using the Macro Recorder Data Explorer ™ So ftw are User ’ s Guide 6-4 5 6 6.7.8 Running Macros Automatically When Opening and Closing Files Y o u can set the Data Explor er software to automati cally ru n macro s you prev iously cre ated when y ou open o r close a data file.
Chapter 6 Using T ools an d A pplicati ons 6- 46 Applied Biosystems 6.
7 Chapter Data Explorer ™ So ftw are User ’ s Guide 7 -1 7 Dat a Explorer Examples This chapter contains the following sections: 7.1 Mariner Data Examples ........... ....................... .... 7-2 7.1.1 Improving Signal-T o-Noise Ratio ........
Chapter 7 Data Explor er Examples 7- 2 Applied Biosystems 7 7.1 Mariner Data Example s This se ction incl udes: • Improv ing sign al-to-nois e ra tio • Deconvo luting an d evaluat ing unres olved chroma tographic p eaks • Determi ning if a peak is b ackground noise 7.
Mariner Data Exampl es Data Explorer ™ So ftw are User ’ s Guide 7 -3 7 Figure 7-1 i llustrates the improved signal-to-noise ratio in the extracted ion chromatogram for thr ee replicate loop injections.
Chapter 7 Data Explor er Examples 7- 4 Applied Biosystems 7 7.1.2 Deco nvoluting a nd Evaluating Unre solved Chromatographic Peaks Overv iew Y ou can use t he Data Explo rer software t o deconv olute .
Mariner Data Exampl es Data Explorer ™ So ftw are User ’ s Guide 7 -5 7 The combined spectrum is displayed (Figure 7-3), with two intense peaks at 410 Da and 723 Da. Generate extracted ion chromatograms as described below to determine if these peaks are the coeluting components.
Chapter 7 Data Explor er Examples 7- 6 Applied Biosystems 7 Figure 7-4 De con voluting Unresolved Chromatographic Peaks Creating combine d spectra Cr eate a com bined spec trum for each e xtracted i on chroma togram: 1. Activate the Spectrum window , then click in the toolbar two times to add two traces.
Mariner Data Exampl es Data Explorer ™ So ftw are User ’ s Guide 7 -7 7 Figure 7-5 illustrates the c ombined spec tra for the deconv oluted pe aks. Note that both spec tra contain a pe ak at 391 Da which requir es invest igation to determin e if it is a low-leve l compon ent or back ground no ise.
Chapter 7 Data Explor er Examples 7- 8 Applied Biosystems 7 7.1.3 Determining if a Peak is Background Noise Overv iew T o deter mine if spe ctral peak s represe nt low-leve l componen ts or if they ar.
Mariner Data Exampl es Data Explorer ™ So ftw are User ’ s Guide 7 -9 7 The spectrum r ange i s displayed i n the Spectr a T o Be Subtracted list in the Add and Subtract Spectra dialog box (Figure 7-6) . Figure 7-6 Subtracting Spectra 6. Click OK .
Chapter 7 Data Explor er Examples 7- 10 Applied Biosystems 7 Figure 7-7 Subtracted Spectrum The peak at 391 is s till pres ent, which in dicates one of the followin g conditi ons: • Y o u did not s .
Voyager Data Exampl es Data Explorer ™ So ftw are User ’ s Guide 7-1 1 7 7.2 Voyager Data Examples This sect ion incl udes: • Detecting and lab el ing partiall y resolv ed peaks • Processi ng before calibrati ng to opti mize mass accu racy • Detecting peak s from co mpl ex diges ts 7.
Chapter 7 Data Explor er Examples 7- 12 Applied Biosystems 7 Figure 7-9 Partially Resolved Peaks That Rep resent Two C ompounds, Minor Component Not Detected Adjusting peak detection T o adjust p eak detec tion: 1. Click in the toolbar or select Peak Detection from the Peaks menu.
Voyager Data Exampl es Data Explorer ™ So ftw are User ’ s Guide 7-1 3 7 2. D o either o f the followin g: • Change the %Max Peak Area from 1 ( the default) to 0 , and the %Base Peak Intensity from 0 (the default) to 1 .
Chapter 7 Data Explor er Examples 7- 14 Applied Biosystems 7 7.2.2 Processing Before Calibrating to Optimize Mass Accuracy This se ction incl udes: • Calibr ating witho ut baseli ne correcti ng and .
Voyager Data Exampl es Data Explorer ™ So ftw are User ’ s Guide 7-1 5 7 Before calibra ting T o opti mize mas s accura cy , do the following befo re cali brating: 1. Display the spectrum of interest. Baseline corre cting 2. From the Proces s menu, sel ect Baseline Co rrection .
Chapter 7 Data Explor er Examples 7- 16 Applied Biosystems 7 Calibrating T o cali brate the dei sotoped s pectrum: 1. From the Peaks menu, select Peak Label , and select the Mass Label T ype (peak apex or peak centroid) to use for calibration. Click OK .
Voyager Data Exampl es Data Explorer ™ So ftw are User ’ s Guide 7-1 7 7 Matching peaks 5. Click M a tch Peaks and S ol ve . The softwar e compares obser ved masses in th e spectrum to reference m.
Chapter 7 Data Explor er Examples 7- 18 Applied Biosystems 7 7.2.3 Detecting Peaks from Complex Digest s Overv iew Comple x dige sts often co ntain hundreds of peaks whi ch may have rel atively low si gnal-to -noise rati os. T o qu ickly scre en out noise an d detect p eaks of inter est: • Nois e filter/sm ooth to re move initia l noise.
Voyager Data Exampl es Data Explorer ™ So ftw are User ’ s Guide 7-1 9 7 Procedure T o d etect peaks from complex mi xtures: 1. Display the spectrum of interest. Noise filteri ng/sm oothing 2. From the Process men u, select Noise Filt er/Smooth . The Noise Filter/Smooth dialog box (Figure 7-19) is displayed.
Chapter 7 Data Explor er Examples 7- 20 Applied Biosystems 7 Figure 7-20 Sp ectrum Peak Det ection Setup — Basic Settings T ab 5. Cli ck U se Advanced Settings .
Voyager Data Exampl es Data Explorer ™ So ftw are User ’ s Guide 7-2 1 7 Figure 7-21 Spectrum Peak Detection Setup — Advanced Settings Tab 6. S et Min im um Area to 0 . 7. Click t he Basic Settings t ab ( se e F igure 7 -20 on page 7-20 ) , then s et %Max Peak Area to 0 or 0.
Chapter 7 Data Explor er Examples 7- 22 Applied Biosystems 7 Figure 7-22 Deisoto ping Dialog Box 10. For this example spectrum, specify H for Adduct and C6H5NO for Generic Formula. 1 1. Click OK . For more informat i on on deisotoping, see Section 3.4, Deisotoping a Spectrum.
8 Chapter Data Explorer ™ So ftw are User ’ s Guide 8 -1 8 V iewing V oyager PSD Dat a This chapter contains the following sections: 8.1 Displaying PSD Dat a ............... ....................... . 8-2 8.2 Applying Fragment Label s .............
Chapter 8 Viewing Voyager PSD Data 8- 2 Applied Biosystems 8 8.1 Displaying PSD Data This se ction incl udes: • Display ing the com posite s pectrum • Advanc ing through segment tr aces • Displa.
Displaying PSD Data Data Explorer ™ So ftw are User ’ s Guide 8 -3 8 Figure 8-1 PSD Spectrum in Dat a Explorer Advancing through segment traces T o a dvance t hrough segm ent trace s, click and . Segmen ts are display ed in the order in wh ich they were acqu ired.
Chapter 8 Viewing Voyager PSD Data 8- 4 Applied Biosystems 8 The PSD Processing dialog box is displayed (Figure 8-2) and lists all segments cont ained in the PSD .DA T file in the order in which they were acquired with associated Mirror Ratios and Max S titch Masses .
Displaying PSD Data Data Explorer ™ So ftw are User ’ s Guide 8 -5 8 NOTE: The entry number in the PSD Segment list above may not correspond to Segment number specified in the Segment list for acq.
Chapter 8 Viewing Voyager PSD Data 8- 6 Applied Biosystems 8 How the composite spectrum is generated The sof tware does th e followin g to gener ate a compos ite spectrum: • Evalua tes all seg ments in the .D A T fi le to deter mine if there ar e multiple segme nts acquired usi ng the sam e PSD Mirror Ratio.
Displaying PSD Data Data Explorer ™ So ftw are User ’ s Guide 8 -7 8 If yo u are perfo rming an internal s tandard calibrat ion, the softwar e determin es the con stants as listed b elow: Region of segmen ts includ ed in composite spectrum The comp osite spec trum is gen erated from portions of the segmen t traces.
Chapter 8 Viewing Voyager PSD Data 8- 8 Applied Biosystems 8 8.2 Appl ying Frag ment Labe ls Overv iew Use th e Ion Fragm entation cal culator to a pply fragme nt labels. For detailed informati on on usi ng the Ion Fragmentation calculato r , s ee Secti on 6.
Applying Fr agment Labe ls Data Explorer ™ So ftw are User ’ s Guide 8 -9 8 2. I n the Seque nce text box, type the amino ac id sequen ce of the c ompound. Us e single-let ter codes. S et other parameter s as needed . For paramete r descriptions , see Secti on 6.
Chapter 8 Viewing Voyager PSD Data 8- 10 Applied Biosystems 8 8.3 Calibrating a PSD Spectrum NOTE: Multi- point calibration yields higher mass accuracy than one-point calibration. This se ction incl udes: • Checki ng peak de tection • Calib rating • Creatin g PSD .
Calibrating a PSD Spectr um Data Explorer ™ So ftw are User ’ s Guide 8-1 1 8 8.3.1 Checking Peak Detection Checki ng Be fore cali brating, ch eck that peaks in all segmen t traces of intere st are prop erly peak detected and that noise is not detecte d as peaks.
Chapter 8 Viewing Voyager PSD Data 8- 12 Applied Biosystems 8 8.3.2 Calibrating This se ction incl udes: • Calib rating • Matchin g peaks automatic ally • Sele cting pea ks manua lly • Solvi n.
Calibrating a PSD Spectr um Data Explorer ™ So ftw are User ’ s Guide 8-1 3 8 Figure 8-5 PSD Processing Dialog Bo x w ith Calibration T ab Displayed 4. Select a PSD Calibration Reference File that you genera ted as desc ribed in Sect ion 8.3.4, Cre atin g PSD Calibr ation Refer ence (.
Chapter 8 Viewing Voyager PSD Data 8- 14 Applied Biosystems 8 NOTE: If the calibration reference file is stored on a network drive, an error message may display when you select the calibration file when perfor m ing a calibration. If an error message is dis played, copy the file to a loca l drive on your computer using Windows NT Explorer .
Calibrating a PSD Spectr um Data Explorer ™ So ftw are User ’ s Guide 8-1 5 8 Matching peaks automatica lly I f you want th e software to compare obser ved masse s in all segmen t spectra includ ed in the .DA T file to refer ence masses in the s elected calibrati on referenc e file: 1.
Chapter 8 Viewing Voyager PSD Data 8- 16 Applied Biosystems 8 Selecting peaks manually For optim um mass accuracy , sele ct peaks as descr ibed in “ Selecti ng calibra tion peak s for opt imum mas s accurac y ” on page 8-19. T o ma nually s elect th e referenc e mass for a peak: 1.
Calibrating a PSD Spectr um Data Explorer ™ So ftw are User ’ s Guide 8-1 7 8 2. Do any of the following: • Click OK to accept the highlighted reference mass for matching.
Chapter 8 Viewing Voyager PSD Data 8- 18 Applied Biosystems 8 Solv in g a nd plotting After matchin g peak s, clic k Solve and Plot . The cal ibration s tatistics are di splaye d in the Resul t tab of.
Calibrating a PSD Spectr um Data Explorer ™ So ftw are User ’ s Guide 8-1 9 8 Selecti ng calibration peaks for optimum mass accuracy T o imp rove cali bration statistics, you can se lect the same fragme nt ion from m ore than one s egment. M onoamino a cid fragme nts (immonium ions) belo w 150 Da ar e useful for this purpos e.
Chapter 8 Viewing Voyager PSD Data 8- 20 Applied Biosystems 8 8.3.3 Creating PSD Ca libration (.CAL) Files and Applyin g to Other Dat a Files Creating P SD .CAL files T o ge nerate a PSD .CAL file: 1. Acquire a standard, for example, angiotensin, in the Instrument Control Panel in PSD mode.
Calibrating a PSD Spectr um Data Explorer ™ So ftw are User ’ s Guide 8-2 1 8 8.3.4 Creating PSD Calibration Referenc e (.REF) Files Overview Y o u can manu ally cre ate a calibrati on referen ce file by ty ping masse s in a text file as desc ribed in Section 5.
Chapter 8 Viewing Voyager PSD Data 8- 22 Applied Biosystems 8 NOTE: This selec tion deter mines the mass type specifi ed for the refe rence mass es in the c alibration reference fil e. Use a calibration refer ence (.REF) file that specifies the peak type for reference masses as Resolved Isotope Mass (even if they are not resolved isotopes).
Calibrating a PSD Spectr um Data Explorer ™ So ftw are User ’ s Guide 8-2 3 8 8.3.5 Changing the Precursor Mass When to ch ange precursor mass When an alyzing th e compos ite spec trum, you ma y find tha t the o bserved frag ments and s equence are not consis tent with the pre cursor mass u sed to acquire the .
Chapter 8 Viewing Voyager PSD Data 8- 24 Applied Biosystems 8 Changing If the pre cursor mass take n from the data fil e is not co rrect: 1. Display the Segments t ab (see Figure 8-2 on page 8- 4) by doing either of the following: • In the PSD Calibration dialog box, click the Segments tab • From the Process menu, select PSD Processing 2.
9 Chapter Data Explorer ™ So ftw are User ’ s Guide 9 -1 9 T roubleshooting This chapter contains the following sections: 9.1 Overview ...................... ................. .................. 9-2 9.2 General Troubleshooting................ ....
Chapter 9 T roub leshoot ing 9- 2 Applied Biosystems 9 9.1 Ov erview This se ction incl udes: • Gener al troubles hooting • Proce ssin g, to ol s, and appli ca tion s troubleshoo tin g • Calibr .
General Trouble shootin g Data Explorer ™ So ftw are User ’ s Guide 9 -3 9 9.2 General Tr oubles hooting T ab le 9-1 Gen eral Trouble shoo tin g — Mariner and Voyager Symptom Po ssible Cause Action Cannot find data file Did not save the spectrum to a .
Chapter 9 T roub leshoot ing 9- 4 Applied Biosystems 9 M/z range in dat a files converted to centroid does not match m/z range in original data f i le M/z range in a dat a file that is converted from profile to centroid is determined by the peak detection range set in Data Explorer , not the m/z range in the original data file No action.
General Trouble shootin g Data Explorer ™ So ftw are User ’ s Guide 9 -5 9 Spectra labeled with spectrum numbers that do not correspond to the axis Y ou are viewing event-filtered MS Method data. Spectra in an event-filtered trace are numbered contiguously (1,2,3.
Chapter 9 T roub leshoot ing 9- 6 Applied Biosystems 9 9.3 P rocessi ng, To ols, and Applic ations Tr oubleshoo ting T able 9-4 Processing , T ools, and Applications Troubleshootin g — Mariner and V.
Processing, Tools, an d Applicati ons T r ouble shootin g Data Explorer ™ So ftw are User ’ s Guide 9 -7 9 Results not saved for all traces in an overlaid trace Only re sult s for t he act ive trace are saved Display individual traces, then save results for each trace.
Chapter 9 T roub leshoot ing 9- 8 Applied Biosystems 9 After Single-charge Conversion of multiply charged peaks, you see charge states other than 0 or 1 Peaks in the original spectrum are labeled with an incorrect charge state 1. Set peak detection thresholds to disregard these peaks.
Processing, Tools, an d Applicati ons T r ouble shootin g Data Explorer ™ So ftw are User ’ s Guide 9 -9 9 T able 9-5 Processing, T ools, and Applications T roubleshooting — Mariner Only Symptom.
Chapter 9 T roub leshoot ing 9- 10 Applied Biosystems 9 9.4 C alibrat ion Tro ubleshoot ing T able 9 -6 Calibrat ion Trou bleshooti ng — Mar iner and V oyager Symptom Possible Ca use Action Auto Cal.
Calibrati on Tr ouble shootin g Data Explorer ™ So ftw are User ’ s Guide 9-1 1 9 Calibration returns an invalid number of matches When creating a reference mass list in Manual or Automatic calibr.
Chapter 9 T roub leshoot ing 9- 12 Applied Biosystems 9 T ab le 9-7 C ali brat ion Trou blesh ooti ng — Mariner Only Symptom Possible Ca use Action Mass Calibration commands are dimmed Chromatogr am window is selected Select Spectrum window .
Calibrati on Tr ouble shootin g Data Explorer ™ So ftw are User ’ s Guide 9-1 3 9 Table 9-8 Calibr ati on Troubl esh ooti ng — Voyager Only Symptom Po ssible Cause Action Error displayed when you import a cal ibration .CAL file corrupted Create new .
Chapter 9 T roub leshoot ing 9- 14 Applied Biosystems 9 9.5 P rinting Troubles hooting T able 9 -9 Print ing Trou bleshootin g — Mariner and V oyager Symptom Possible Ca use Action T races do not print Line wid th is set to 0 or 1 Change the line width.
Peak Detection and Labeling Tr oubleshootin g Data Explorer ™ So ftw are User ’ s Guide 9-1 5 9 9.6 Peak Dete ction an d Labeli ng Trou blesho oting This sect ion incl udes: • Peak dete ction an.
Chapter 9 T roub leshoot ing 9- 16 Applied Biosystems 9 Peaks are not detected or labeled (continued) Analyzing masses above 20,000 Da Increase Mass Resolution setting in Peak Detection Setup. See Section 3.2.2 , S tr ategy for V oyager Peak Detection.
Peak Detection and Labeling Tr oubleshootin g Data Explorer ™ So ftw are User ’ s Guide 9-1 7 9 Expected user label not displayed Delta X value includes more than one peak apex Set Delta X value low enough to prevent the peak labeling windows from overlapping.
Chapter 9 T roub leshoot ing 9- 18 Applied Biosystems 9 Partially resolved peaks not detected Mass resolution set too high to detect average mass Decrease Mass Resolution setting. See Section 3.2.2, S trategy for V oyager Peak Detection. %Base Peak Intensity not adjusted correctly Adjust.
Peak Detection and Labeling Tr oubleshootin g Data Explorer ™ So ftw are User ’ s Guide 9-1 9 9 Known isotope not labeled with charge state Charge S tate peak labels disabled Tur n on Charge S tate peak labels. See Section 3.5.2, Setting Chromatogram and Spectrum Peak Labels.
Chapter 9 T roub leshoot ing 9- 20 Applied Biosystems 9 Spectrum peaks not labeled with charge stat e when charge state labels are selected Mass of original molecule above 4,000 Da, not range in which the Mariner system can r esolv e the isotope peaks No action.
Peak Detection and Labeling Tr oubleshootin g Data Explorer ™ So ftw are User ’ s Guide 9-2 1 9 Spectrum peaks labeled with incorrect charge state when charge state labels are selected (continued) Charge state determination par ameters are set such that peaks are determined to have no cha rge Adjust parameters.
Chapter 9 T roub leshoot ing 9- 22 Applied Biosystems 9.
A Appendix Data Explorer ™ So ftw are User ’ s Guide A- 1 A W arranty Applied Biosystems supplies or recommends cert ain configurations of computer hardware, softw a re, and peripherals for use with its instru ment ation.
Appendix A Warranty A- 2 Applied Bio systems A not warrant that th e operation of the i nstrument o r software will be uninter rupted or error free. Applied Biosystem s will provi de any so ftwar e correc tions or “ bug- fixes ” , if an d when they become av ailable , for a peri od of ninet y (90) days after installatio n.
Data Explorer ™ Softwar e User ’ s Guide A -3 A W arranties limitations THE REMEDI ES PROVIDED HEREIN ARE B U YER ’ S SOLE AND EXCLUS IVE REMEDIES.
Appendix A Warranty A- 4 Applied Bio systems A.
B Appendix Data Explorer ™ So ftw are User ’ s Guide B- 1 B Overview of I sotopes This appendix con tains the following sections: B.1 Isotopes ...................... ...................... ........ B-2 B.2 Monoisotopic and A verage Mass es .......
Appendix B Overvi ew of I sotope s B- 2 Applied Bio systems B B.1 Is otopes Overvi ew Man y elements in th eir natura l state exist as one of se veral isotopes . An isotope is one of two or more atoms with the same a tomic number but a differen t mass.
Isotopes Data Explorer ™ Softwar e User ’ s Guide B -3 B As the numbe r of carbon atoms in a com pound increas es, the possi bility of th e compoun d containing a 13 C inste ad of a 12 C also inc reases.
Appendix B Overvi ew of I sotope s B- 4 Applied Bio systems B Figure B-3 Mass Spectrum o f Angiotensin I at Resolution 3,000 In compoun ds with mo re than 100 c arbon atom s, the height o f the first 13 C isotope p eak exceeds the heig ht of the 12 C peak.
Isotopes Data Explorer ™ Softwar e User ’ s Guide B -5 B Figure B-4 Mass Spectrum of A ngiotensin I at Resolution 1,000 If isoto pes cann ot be resol ved, the hi ghest reso lution you can obtain is limited by t he width of the i sotopic env elope.
Appendix B Overvi ew of I sotope s B- 6 Applied Bio systems B B.2 Monoiso topic and Aver age Masse s When iso topes ar e clearly r esolved ( Figure B-6), the monoisot opic mas s is used for mass labelin g, and correspo nds to the low est mass peak in the cl us ter .
Monoisotopic and A verage Masses Data Explorer ™ Softwar e User ’ s Guide B -7 B Figure B-7 Average Mass A v erage mass corresponds to centroid of unresolved peak cluster.
Appendix B Overvi ew of I sotope s B- 8 Applied Bio systems B B.3 I sotopes of Common Elements T able B-1 li sts the natural abundanc e of isoto pes for som e commo n elements seen in mass sp ectrome try T able B-1 Isoto pes of Common Elements 1 Isoto pe Mass Nat ural abunda nce (%) Isot ope Mass N atural abundance (%) 1 H 1.
C Appendix Data Explorer ™ So ftw are User ’ s Guide C -1 C Dat a Ex plorer T oolbox (V isual Basic Macros) This appendix includes: C.1 Overview ........................ ................. ....... C-2 C.2 Pr eparing Data Before Access i ng Macros .
Appendi x C Data Explo rer T ool box (Visual Bas ic Macros) C- 2 A ppli ed Biosystems C C.1 Ov erview Macros provided The foll owing tool box of Visual B asic mac ros is prov ided with the Data Expl o.
Preparing Dat a Befor e Acce ssing Macr os Data Explorer ™ So ftw are User ’ s Guide C -3 C References required The followin g referenc es are sel ected by def ault in the DataExplorer Project in the Visual Ba sic Edito r and ar e requi red for the m acros in t he DataExplorer.
Appendi x C Data Explo rer T ool box (Visual Bas ic Macros) C- 4 A ppli ed Biosystems C C.3 Access ing the Macr os T o access the macros : 1. Open the Data E xplorer software. 2. O pen a data file . 3. Pr epare the data as d escribed i n the pre vious sectio n.
Using the L adder Sequencing Toolbox Data Explorer ™ So ftw are User ’ s Guide C -5 C NOTE: If the modT oolBoxPalette.T oolbox_Palette is not listed, you must import the macro into t he Data Explorer project. For i nformation, see S ection 6.7.7, Importing or Exporting Macros in DA T AEXPLORER .
Appendi x C Data Explo rer T ool box (Visual Bas ic Macros) C- 6 A ppli ed Biosystems C 2. En ter the ma ss toleran ce to appl y to the a nalysis. 3. Cli ck G et Spec Peak List . 4. Rem ove peaks you do not want included in the calculation by click ing the peak in the li st, then cli cking Delete Sel ected Peaks /Add uct s .
Using the L adder Sequencing Toolbox Data Explorer ™ So ftw are User ’ s Guide C -7 C 7. U nder Anno tate Spectrum, s elect th e types of la bels yo u want d isplayed: • Reference Mass (*) Mass of the r eference peak against which the current peak is compared.
Appendi x C Data Explo rer T ool box (Visual Bas ic Macros) C- 8 A ppli ed Biosystems C If DNA or RNA is s el ected, the softwar e: • Examines the spectrum in 270 Da increment s and selects the most intense ion in the range. (The 270 Da increment is used because it is less than the smallest mass difference related to a DNA or RNA base.
Using the P eptid e Fragment ation Toolbox Data Explorer ™ So ftw are User ’ s Guide C -9 C C.5 Using the Peptide Frag mentation T oolbox Use the Peptide F ragmentatio n toolbox whe n examini ng V.
Appendi x C Data Explo rer T ool box (Visual Bas ic Macros) C- 10 Applied Biosystems C 3. Ad d peaks to t he peak list to be incl uded in the cal culation by typi ng a mass in the Add M ass to Pe ak List fie ld, then clicki ng Add Peak .
Using the P eptid e Fragment ation Toolbox Data Explorer ™ So ftw are User ’ s Guide C -11 C • y and b p airs Lists peak pairs whose combined masses plus 1 Da add up to the Precursor Ion Mass you specified on the Setup tab. • Loss of H2O Lists peak pairs with a 18 D a mass difference.
Appendi x C Data Explo rer T ool box (Visual Bas ic Macros) C- 12 Applied Biosystems C 2. If d esired, click Label Immonium Ions . NOTE: Label immonium ions before selecting a reference peak and st arting the search.
Using the P eptid e Fragment ation Toolbox Data Explorer ™ So ftw are User ’ s Guide C -13 C 5. Click Copy to Output Window Result T ab to copy results to the Resu lt tab. Y ou c an then co py from the Resu lt tab to anothe r applicat ion such as Notepad , or print, as needed.
Appendi x C Data Explo rer T ool box (Visual Bas ic Macros) C- 14 Applied Biosystems C 4. Cli ck Find C orrelation . Correlations for the selected peak are listed. NOTE: Y p, Sp and Tp represent phosphotyrosine, phosphoserine, and phosphothreonine, respectively .
Using the Polymer Analysis Toolbox Data Explorer ™ So ftw are User ’ s Guide C -15 C C.6 Using t he Polyme r Analy sis Toolbo x Use the Polymer Anal ysi s T oolbox to deter mi ne the fol lowing va.
Appendi x C Data Explo rer T ool box (Visual Bas ic Macros) C- 16 Applied Biosystems C 3. Se lect the m ode for th e analys is: • Use the entire mass range — Calculates average molecular weights using all peak intensities within the X Display Range.
Using the Polymer Analysis Toolbox Data Explorer ™ So ftw are User ’ s Guide C -17 C • Use labeled peaks — Calculates average molecular weights using areas of peaks listed in the Spec Peak List. It allows calculation of values for a distinct polymer series when two or more species are present.
Appendi x C Data Explo rer T ool box (Visual Bas ic Macros) C- 18 Applied Biosystems C C.7 Using MS Fit/MS Ta g Toolbo x Use the MS Fit/MS T ag toolbox when analy zing pro tein digest , peptide, o r peptide fragment spectra to perform a pr otein database sear ch.
Using MS Fi t/MS Tag Toolb ox Data Explorer ™ So ftw are User ’ s Guide C -19 C 2. Click: • MS-Fit tab If you are examining peptide dat a from a protein digest. • MS-T ag tab If you are examining PSD dat a. 3. N avigate to the web si te containin g the database to search .
Appendi x C Data Explo rer T ool box (Visual Bas ic Macros) C- 20 Applied Biosystems C.
Data Explor er Sof tware U ser ’ s G u i d e In dex-1 Inde x I D E X N Numerics and Sym bols - in spectrum header 2-31 %Base Peak Intensity definition, chromatogram 3-20 definition, spectrum 3-22 in.
I D E X N Index-2 Ap p l i e d Bi o s y st em s Amino acids, labeling C-5 Analog signal, displaying 4-2 Analyzer T emperature, displaying trace 4-2 ANGIOTENSIN_FR AGMENTS.
Data Explor er Sof tware U ser ’ s G u i d e In dex-3 I D E X N Bar mode, traces 1-28 BAS files for macros 6-43 Base mass, labeling peaks 3-55, 3- 56 Base peak intensity definition 4-2 scaling to 2-.
I D E X N Index-4 Ap p l i e d Bi o s y st em s Calibrating mass, automatic (Mariner data only) ( c ontinued) reverting to instrument calibration 5-22 troubleshooting 9-9 turning on 5-34 when to use 5.
Data Explor er Sof tware U ser ’ s G u i d e In dex-5 I D E X N Calibration constant s see also Calibrating mass applying to new file 5-16, 8-20 calculating 5-34 displayed in Output window 5-14, 8-1.
I D E X N Index-6 Ap p l i e d Bi o s y st em s Charge stat e, peak (continued) requirements for labeling 3-53 single 5-59 tolerance calculation 3-32 troubleshooting 9-17 z labels 3-58 zero 3-40, 3-43.
Data Explor er Sof tware U ser ’ s G u i d e In dex-7 I D E X N CombiSolv data displaying one injection 4-24 Event T ag Filtering command dimmed (Mariner data only) 4-24 Comment acquisition, display.
I D E X N Index-8 Ap p l i e d Bi o s y st em s Customizing (continued) SET files 1- 17 toolbars 1-22 D DAD displaying Channels 1- 12, 4-2 displaying T AC 1- 12 displaying traces 2-6 extracted absorba.
Data Explor er Sof tware U ser ’ s G u i d e In dex-9 I D E X N Data file (cont i nued) full name not displayed 1- 14 inserting traces into 2-37 moving between open 2- 8 multiple, zooming 2-36 name .
I D E X N Index-10 A p p l i e d Bi o sy st e m s Detection Ranges (continued) setting manually , chromatogram 3-19, 3-20 setting manually , spectrum 3-28 setting parameters globally , spectrum 3-22 s.
Data Explor er Sof tware U ser ’ s G u i d e Index-11 I D E X N Excel, see M icrosoft Excel Exiting software 1-3 Expanding traces 2-21 Exporting see also Converting ASCII data 1-34 BIC 1-36 CAL files 1-36 Configuration from DA T file 1-36 entire data file 1-34 macros from DA T A EXPLORER.
I D E X N Index-12 A p p l i e d Bi o sy st e m s Filter Width I nc rement setting manually , spectrum 3-31 suggested value, spectrum 3-31 value used when resolution-based peak detection enabled 3-31 .
Data Explor er Sof tware U ser ’ s G u i d e Index-13 I D E X N I Immonium ions, labeling C-9 IMMONIUM_IONS.REF 5- 18, 8-19 Import Calibration error displayed 9-1 1, 9-12 procedure 5-16 PSD 8-20 Importing macros into DA T A EXPLORER.
I D E X N Index-14 A p p l i e d Bi o sy st e m s Isotope Match Intensity , elemental targeting 6 -33 Isotope Match Score elemental composition 6-6 elemental composition, not reported for fragment ion.
Data Explor er Sof tware U ser ’ s G u i d e Index-15 I D E X N M Macro Recorder advanced editing 6-42 buttons, assigning to macros 6-38 DA T AEXPLORER.VB6 location 6-35 DA T AEXPLORER.VB6 not overwritten when new software installed 6-43 deleting a macro 6- 41 description 6-34 exporting macros from DA T A EXPLORER.
I D E X N Index-16 A p p l i e d Bi o sy st e m s Mass Calibration commands dimmed on menu 9-1 1 not displayed on menu 9-8 Mass calibration, see Calibrating mass Mass, centroid calculating 3-39 copyin.
Data Explor er Sof tware U ser ’ s G u i d e Index-17 I D E X N MS Method (Mariner dat a only) (continued) instrument settings, viewing 4-23 spectrum numbers in filtered trace 4-25 MSM files extract.
I D E X N Index-18 A p p l i e d Bi o sy st e m s Output window acquisition comment, displaying 1- 15 calibration statistics, displaying 5-13, 8-18 Chr o P ea k lis t ta b 1- 1 5 clearing 1- 16 closin.
Data Explor er Sof tware U ser ’ s G u i d e Index-19 I D E X N Peak detection (continued) Noise Threshold, calculated automatically for chromatogram data 3- 21, 3-68 overview 3-2 Peak Processing pa.
I D E X N Index-20 A p p l i e d Bi o sy st e m s Peak labels (continued) centroid 3-56 chromatogram, setting 3-54 custom 3-61 custom, creating for fragment spectra 8-9 customizing 1-25 decimal places.
Data Explor er Sof tware U ser ’ s G u i d e Index-21 I D E X N Peak weighting factors 5-1 0, 8-14 Peak Width minimum and maximum used 3-21, 3-25 set automatically by software 3-21, 3-25 Peaks, do n.
I D E X N Index-22 A p p l i e d Bi o sy st e m s PSD analysis (continued) composite spectrum, displaying 8-2, 8-5 composite spectrum, how it is generated 8-6 fragment labels, applying 8-8 optimum res.
Data Explor er Sof tware U ser ’ s G u i d e Index-23 I D E X N Result tab, O utput window 1- 15 Results see also RSD and RCD annotating tr aces with 2-28 copying 2-28 displaying in Output window 1-.
I D E X N Index-24 A p p l i e d Bi o sy st e m s Sequence Control Panel, Mariner , automatic calibration settings (reference masses) for 5-27 Sequence Control Panel, V oyager , automatic calibration .
Data Explor er Sof tware U ser ’ s G u i d e Index-25 I D E X N Spectra (continued) summing non-contiguous 4-21 troubleshooting 9-17 truncating 5-56 types of 5-2 Spectrum noise threshold, setting lo.
I D E X N Index-26 A p p l i e d Bi o sy st e m s T T abs for open files 2-8 in Data Explorer window 2-8 TA C in chromatogram header 2-30 Mariner dat a, optional 1- 12 T ag, see Event tag T arget comp.
Data Explor er Sof tware U ser ’ s G u i d e Index-27 I D E X N T races (continued) Replace mode, setting 2-17 scaling mode, setting 2-12 splitting 2-15 switching between 2-8 text, customizing 1-25 .
I D E X N Index-28 A p p l i e d Bi o sy st e m s V V alley-to-Baseline integration chromatogram 3-21, 3-70 spectrum 3-26, 3-70 V alley-to-V alley i ntegration chromatogram 3-21, 3-70 spectrum 3-26, 3.
Data Explor er Sof tware U ser ’ s G u i d e Index-29 I D E X N X X cursors, setting 1-27 x,y data p airs, copying 1-39 XAC in chromatogram header 2-30 see also Extracted absorbance chromatogram (XA.
I D E X N Index-30 A p p l i e d Bi o sy st e m s.
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