An Scripts Guide en
An Scripts Guide en
2 Software
RUO-IDV-05-0270-A
Release Date: April 2013
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Contents
Foreword. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Analyst Software Scripts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Install or Uninstall Scripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Add Missing Zeros . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Add Normalized ADC Traces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Analyst 1.2 Peak Finder Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Batch Script Driver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Change All Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Convert Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Create Quantitation Methods and Text Files . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
DBS Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Define Custom Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
Delete Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
DFT Tracker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Export IDA Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
Export Sample Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
Export to JCamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
IDA Trace Extractor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
Label Selections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40
Label XIC Traces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
Make Exclusion List from Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
Make Subset File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43
Manually Integrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
Mascot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48
Mass Defect Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
Merge MRM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54
MRM3 Optimization Script . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55
MS3 Quant Optimization Script . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65
Multiple Batch Scripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66
Open in Workspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67
Peak List from Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68
Regression Calculator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69
Remove Graph Selections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70
Repeat IDA Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
Savitzky-Golay Smooth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .72
Selection Average and Standard Deviation . . . . . . . . . . . . . . . . . . . . . . . . . . . .73
Send to ACD SpecManager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74
Signal-to-Noise Using Peak-to-Peak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76
Signal-to-Noise Using Standard Deviation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
Split Graph Script . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78
Subtract Control Data from Sample Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79
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Contents
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Foreword
This guide provides information on how to use the Analyst® software scripts and is intended for
customers and FSEs.
Related Documentation
The guides and tutorials for the mass spectrometer and the Analyst® software are installed
automatically with the software and are available from the Start menu: All Programs > AB SCIEX
> Analyst. A complete list of the available documentation can be found in the online Help. To view
the Analyst software Help, press F1.
Technical Support
AB SCIEX and its representatives maintain a staff of fully-trained service and technical
specialists located throughout the world. They can answer questions about the system or any
technical issues that might arise. For more information, visit the Web site at www.absciex.com.
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Foreword
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Analyst Software Scripts
The purpose of this document is to explain how to install and use Analyst® software scripts. It
also provides an overview of the uses of each script and how to uninstall a script, if required.
Related Documentation
You can also create your own scripts using the Visual Basic Version 6 program. For more
information, refer to the Analyst® Automation Cookbook: A guide to controlling Analyst® Software
from Visual Basic .NET.
Note: This guide contains the scripts for all instruments and different software
versions. To determine which scripts the software version installed on your instrument
supports, refer to the Analyst software release notes.
Install a Script
1. Navigate to the following folder on your workstation: <drive>:\Program
Files\Analyst\Scripts.
2. Open the required folder, open the script folder, and then double-click
ScriptRunner.exe.
3. Follow the on-screen instructions to install the scripts.
The script is available on the Script menu.
Note: For some scripts, if you hold down the Shift key while accessing a
script on the Script menu, a description of the script is displayed.
Uninstall a Script
• To uninstall a script, do one of the following:
• For processing scripts, navigate to the <drive>:\Analyst Data\Projects\API
Instrument\Processing Scripts folder and then delete the script .dll or, if
applicable, .exe and .bmp files, manually.
• For acquisition scripts, navigate to the <drive>:\Analyst Data\Projects\API
Instrument\Acquisition Scripts folder and then delete the script .dll or, if
applicable, .exe and .bmp files, manually.
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Notes
• If the data in the active pane came from several samples, then the ADC data for the
sample corresponding to the first data set (not the active data set) is shown.
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7. To run the script on each data file in the list, click Run.
8. To stop the script, click Close.
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Note: This script is used with any QTRAP® system. It is not used by QSTAR®
instruments.
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Convert Methods
Use this script to convert methods from one type of instrument to another. The script converts the
method to the currently active hardware profile, using appropriate values for each parameter.
Only the ion source and compound dependent parameters for mass ranges and experiments are
shown.
The Convert Methods script automatically optimizes mass ranges and, in addition to single
period, single experiment methods, converts multiple periods, multiple experiments, and IDA
criteria.
Prerequisites
• .Net framework 3.5 SP2 (will be automatically installed if required)
2. Click Open, navigate to the method that you want to convert, and then click Open.
The Method Converter dialog displays the instrument name of the original method.
3. Click Save, type a name for the converted method, and then click Save.
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3. Select the Use Baseline Subtraction check box to have the Window Summation
algorithm sum the intensities to the horizontal line at the minimum intensity of the
data points within the summation window, as opposed to summing down to the
intensity zero.
4. In the Regression Parameters group, select the regression information. The
information specified here is applied to every analyte peak. Unlike the previous
parameters, it is not possible to indicate this information in the text files; therefore,
the same regression parameters are applied to all analytes. For a full description of
the parameters, refer to the Help.
5. To create one quantitation method, click Create One Method and then navigate to
the text file that will be used to create the quantitation method.
6. To create multiple methods from multiple text files, click Create Multiple Methods
and then navigate to the folder. A quantitation method is created for each text file in
this folder.
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3. Select the Export all columns check box and then click OK.
4. Navigate to and select the quantitation method file (.qmf).
5. Navigate to and select the location of the text file.
The script will generate the text file.
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The following table shows an example text file for full-scan data. Imagine that there are tabs
between the columns and a hard return at the end of each line.
Table 1-2 Example Text File for Full-Scan Data
Peak Name First Mass Second Mass Bunching Factor
Analyte Peak 1 500.10 500.70 1
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The following table shows one more example for MRM data. The Analyte Peak 1 will be set up to
use the specified internal standard and Analyte Peak 2 will not use an internal standard.
Table 1-3 Example Text File for MRM Data
Peak Name Is IS IS Name First Mass Second Mass
IS Peak 1 Yes 500.10 413.20
Analyte Peak 1 No IS Peak 1 600.20 382.10
Analyte Peak 2 No 400.00 312.1
The following table contains a mixture of full-scan and MRM data in different experiments:
Table 1-4 Example Text File for MRM Data
Peak Name Extraction Experiment First Mass Second Mass
Type
Analyte Peak 1 0 1 500.10 413.20
Analyte Peak 2 0 1 600.20 382.10
Analyte Peak 3 2 2 812.00 813.00
Analyte Peak 4 2 2 400.00 401.00
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DBS Settings
The DBS (Dynamic Background Subtraction™ algorithm) is a feature that ensures better
selection of precursor ions in an IDA (Information Dependant Acquisition) experiment. When
DBS is activated, IDA uses a spectrum that has been background subtracted to select the
candidate ion of interest for MS/MS analysis, as opposed to selecting the precursor from the
survey spectrum directly. DBS enables detection of species as their signal increases in intensity,
thus focusing on detection and analysis of the precursor ions on the rising portion of the LC peak,
up to the top of the LC peaks (maximum intensity).
The DBS functionality is embedded in the Analyst® software for IDA experiments. However, the
associated parameters are not accessible in the Analyst software. The hidden parameters and
their default values are as follows:
• Average number of previous spectra = 4
• Smooth before subtract: activated
• Smooth = 5 data points.
Use this script to change the default parameters to ones that are more representative of the
experimental conditions. Depending on the cycle time and chromatography, the default settings
may result in an obvious candidate ion from being omitted for dependent MS/MS analysis or the
same candidate ion may be selected for MS/MS analysis over the entire LC peak. Therefore, this
script will be useful to customers who find that the embedded default values are not appropriate
for their analysis.
After the script is installed, the DBS feature uses the settings in the script. The script will
remember the last settings used.
Prerequisites
• Analyst 1.6.2 Software installed
• Administrator rights on the computer
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2. Although the DBS feature is activated at the method level, the DBS options are set
using the script. From the Analyst software menu, click Script > DBS Settings.
• Subtract the _ previous spectra from current spectrum: Use this field to
select the number of spectra averaged to represent the background signal.
These spectra are taken immediately before the current survey spectrum.
Typical values used are between 2 and 5.
• Smooth Before Subtract: Select to make sure that the current survey
spectrum is smoothed using a Savitzky-Golay smooth before the subtraction
step. Select the number of points to consider in the process. A typical value is
5.
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Prerequisites
The following program is optional:
• Metabolite ID 1.3 for Analyst QS 2.0 software
When the script is launched for the first time, a backup copy of the SAElements.ini file is saved in
the API Instrument folder. If needed, the edited SAElements.ini file in Analyst\bin folder can be
replaced with this file.
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View the Custom Element Symbol, Custom Element Name, and Custom Pattern
• To view the custom pattern in the mass/relative intensity graph, in the Define
Custom Elements dialog, click Show.
The Isotopic Distribution dialog opens.
The total of the individual isotope abundance for an element stored in the element
definition file must be equal to one. Therefore, the abundances entered in the Define
Element Window are rescaled before they are added to the Define Custom Elements
dialog. This Isotopic Distribution dialog cannot be edited. You can zoom in the area
of interest by dragging along the corresponding x- or y-axis region.
The application requires the gen01.wiff example file to display custom pattern. If the
gen01.wiff file is not in the Example folder in the Analyst Data\Projects folder, you will
be prompted to find this file.
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Delete Others
Use this processing script to delete all panes except for the active one.
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DFT Tracker
The Dynamic Fill Time (DFT) Tracker script tracks the DFT settings used during QTRAP®
instrument scans. You can use the script to determine the optimal fill time for linear ion trap (LIT)
mode to obtain high data quality over a wide dynamic range. The DFT Tracker monitors the
following LIT scan types: Enhanced MS (EMS), Enhanced Resolution (ER), Enhanced Product
Ion (EPI), and MS/MS/MS (MS3).
DFT Tracker monitors the dynamic changes occurring during a real-time run.
The system dynamically calculates the time required to fill the linear ion trap. For
abundant compounds, a short fill time reduces the space charge effects by limiting
the number of ions in the ion trap; on the other hand the longer fill time increases
weak signals by allowing the ions to accumulate.
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2. In the Mass tolerance for combining MS/MS spectra field, type the tolerance to be
used to determine if two precursor m/z values should be considered identical. If the
precursors for two sequential product spectra differ by less than this value, the
spectra are added and a single text file is exported.
3. In the MS/MS intensity threshold field, type the threshold that is applied to each
product spectrum after it is centroided. It is assumed that peaks below this threshold
are most likely noise. Type 0 in the field if you do not want to use a threshold.
4. In the Minimum number of MS/MS ions for export field, type the minimum number
of ions that must be present in a product spectrum, after centroiding and
thresholding, in order for a text file to be exported. If a spectrum does not contain the
specified number of ions, it is assumed that the quality of the spectrum is too low to
merit exporting.
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5. To separate fields in the output files with a space character, select the Separate
values in output with a space, not a tab check box; otherwise a tab character is
used. (Certain versions of Sequest require a space delimiter.)
6. To export the text files, click Go.
7. In the Save As dialog, type a location and root file name for the exported text files.
Before being exported to a text file, each of the product spectra is centroided. The
cycle number range and charge state is appended to this file for each exported
spectrum.
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2. Select the check boxes that correspond to the information that you want to export
and then click OK.
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Export to JCamp
You can use the Analyst® software to export graph data to a tab-delimited text file that can be
read by most applications. However, some applications require a more specific format.
With the Export to JCAMP script, you can export graph data in the JCAMP format. The script
works on both chromatograms and spectra. For chromatograms, depending on the number of
selections made, either all the spectral data of the chromatogram is exported, or the averaged
sum of the selected regions is exported. If you are using this script on a single spectrum, then
only that data is exported. This script can also be attached to a batch so that the export occurs
automatically after the sample is acquired.
Table 1-5 shows an overview of the operation of the script. When run interactively the exact
behavior depends on the active Analyst software data.
Table 1-5 Script Operation
Modes Active data Operation
Interactive Spectrum You will be prompted for the name of the JCamp file and
the active spectrum exported to it.
Interactive Chromatogram with You will be prompted for the name of the JCamp file and an
two or more averaged spectrum corresponding to each of the
selections chromatogram’s selections exported to it.
Interactive Chromatogram with You will be prompted for the name of the JCamp file and
one or no selections every spectrum for the run exported to it.
Batch N/A The name of the JCamp file is generated by appending the
sample number to the name of the .wiff file and changing
the extension to jdx. Every spectrum for the run is exported
to the JCamp file. For multiple period/experiment data, a
separate file is exported for each experiment (the period
and experiment numbers are appended to the filename).
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• Save the list of masses and peaks in a format that can be loaded to the
CreateQuantMethodFromText script.
Note: The script is compatible with MRM / MIM IDA data and with the Analyst® 1.6.2
software.
Note: The script supports parallel data processing from positive and negative
experiments. Multiple survey and dependent experiments of any polarity can be used.
Note: The Analyst software version for a specific file can be displayed in the file
properties > comments.
1. With a chromatogram of IDA data open in an active pane, click Script >
IDATraceExtractor.
You can process a full chromatogram or just a selected region (make a selection
before running the script).
Figure 1-13 IDA Trace Extractor Dialog
2. In the Options tab of the script, set the fields as required. For more information, refer
to Table 1-6 Tab and Menu Parameters on page 36.
3. To store the retention times and precursor masses found during processing, click the
Results tab, type a filename, and then select Save Precursor Information.
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4. To review or enter the mass information, click the Neutral Losses or Fragments
tab. Type the neutral losses and fragments as masses or chemical formulas. You
can also specify the polarity of the Neutral Loss or fragment spectrum experiment
where the specified neutral loss or fragment is expected to be found.
Figure 1-15 IDA Trace Extractor Dialog: Neutral Losses Tab
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5. Click Extract to find the survey XIC traces that give the selected neutral loss or
fragment.
6. If the precursor information was saved, the found precursor mass/time data can be
converted to a compatible format using other scripts. To convert the data, click the
Results tab and then select the Results File. View and edit this file as required.
• To make a format for the XICfromTable script, click Make XIC Table. The
Results File will be converted into a file of the same name with the suffix _XIC.
• To make a format for the CreateQuantMethodFromText script on the
Precursor XICs dialog, click Make Quant Input. The Results File will be
converted into a file of the same name with the suffix _Peaks.
7. Click File > Save Settings as. Alternatively, previously saved settings can be used.
Click File > Load Settings to open previously saved settings.
Several functions are available in the Tools menu. You can start processing without switching to a
specific tab.
Table 1-6 Tab and Menu Parameters
Location Parameters Description
Neutral Losses Use Masses Select the required neutral loss(es) as
mass.
Neutral Losses Use Formulas Select the required neutral loss(es) as
formula.
Neutral Losses Start Low mass limit (from mass) for the
neutral loss(es).
Neutral Losses End High mass limit (to mass) for the neutral
loss(es).
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Label Selections
Use this script to add missing labels to the selected peaks in the active graph or to remove them.
The script can be run when a pane containing a spectrum or a chromatogram is active in Explore
mode, and there are one or more selections in the active pane. If neither peak mass (spectrum)
nor peak retention time (chromatogram) is available, the data will be marked with information for
the selection maximum.
Note: Only font type and label color are synchronized with the automatic labels. For
the best performance, synchronize the other font attributes manually in the Appearance
Options dialog.
Note: Labeling spectra with centroid mass/charge state appends just the centroid
mass. Labeling chromatograms with base peak ion mass or base peak ion intensity is
not supported.
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2. In the Exclusion List File Name field, type the name and path of the text file.
3. If required, in the Threshold field, type the threshold that will be applied to the
centroided spectrum so Threshold field that small noise peaks are not included in
the exclusion list. Type 0 in the to not use a threshold.
4. Click Export to export the exclusion list using the specified parameters.
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Section 1: General
This section describes how to select a data file to work on.
1. Click Script > MakeSubsetFile.
Figure 1-18 Make Subset File Dialog
2. By default, all the files in the current project data folder are loaded. To add another
file to this list, click File > Open and then navigate to the file.
3. Click the file.
4. To exit the program, click File > Exit.
Section 2: Transfer
This section describes how to transfer samples from one data file to another.
1. Click the Transfer tab.
All the samples in the current working data file will automatically appear in the
Samples to Transfer list.
2. To exclude a sample from the transfer, select it from the list and then click >>.
The unwanted sample is shown in the Samples to Exclude list.
3. To include an excluded sample in the transfer, select it from the Samples to
Exclude list and then click <<.
4. To exclude all the samples, click the Remove All type the full path and the file name
of the file in which the sample will extract, or navigate to a file. If the file does not
exist, the Make Subset File script will create it.
5. To start the transfer, click Extract.
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Section 3: Unpack
This section describes how to unpack every sample in one data file into its own data file.
1. Click the Unpack tab.
2. In the Destination Directory group, the location of the current working data file is
shown. Use this tree to select the location for the unpacked data files.
3. To create a new directory, right-click the directory tree. You will be prompted to type
the new folder name and set the active folder.
4. In the Output File Name text field, type the output file name. Each sample
unpacked from the working data file will begin with this name followed by the sample
number in parentheses. Do not give an extension to this file (for example, do not
include “.wiff”), because the Make Subset File program will automatically append
this.
5. To begin unpacking the samples, click Extract.
Section 4: Decompose
This section describes how to decompose a sample into different samples.
1. Click the Decompose tab.
2. To use the Make Subset File script default values, select the Use Defaults check
box.
3. To provide threshold values, deselect the Use Defaults check box and then type the
values in the fields.
• The Noise field contains the noise threshold value. This value indicates when
a sub-sample begins and ends. If the intensity value exceeds this threshold,
then it is considered a new sample. After an intensity value falls below this
threshold, the sub-sample is considered complete and it is sent to the file.
• The Width field contains the width threshold value. This value is used to
prevent short periods of loud noise. If noise exceeds the noise threshold value
for a short period of time (that is, have a small width) then without the width
threshold this noise will be considered a new, sample. Only detected samples
with a width that exceeds the width threshold will be exported to the file.
4. In the Decompose To field, type the full path and the file name of the file in which
the sample will be decomposed, or navigate to a file. If this file does not exist, then
the Make Subset File program will create it.
5. To start decomposing the sample, click Extract.
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Manually Integrate
The Analyst® software has various peak-finding algorithms that determine and integrate peaks
and then show the results in the peak list. If required, you can also use the Manually Integrate
script to integrate a selected region of a graph because the software may not have detected the
peak or perhaps because only a portion of the peak is of interest.
Use the script to draw a line on a chromatogram and have the area above the line integrated.
The integrated area is highlighted in the chromatogram, and the calculated area of the region can
be pasted on the graph. The results are displayed in the script window, which can also be added
to the graph or exported to a text file for storage.
2. In the Text File Exporting group, click Select to navigate to a text file.
3. Do one of the following:
• Select Automatically export after each selection to automatically export the
results to the specified text file.
• Click Export Now to export the current results.
4. To remove the last exported results from the text file, click Remove Last Entry.
5. To populate the fields in the Result group, highlight a section of the graph. The
values are calculated using the selected area of the graph.
6. To change the manual integration options, click Options.
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• The Fixed RT width: _ sec option is for MALDI workflows only. If selected, the
total width of the resulting peak is fixed at the specified value and is centered
at the apex retention time.
9. To save changes and return to the Manual Integration 1 dialog, click OK.
10. On the Manual Integration 1 dialog, click Close
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Mascot
Use this script to send either the active spectrum or all product spectra contained in the active
sample, or all samples in the active data file, to the Mascot protein search engine. The script was
co-developed with Matrix Science Limited, the creators of Mascot.
When sending only the active spectrum, the script can work with either MS or MS/MS data; in the
first case a peptide mass fingerprint search is conducted. When sending all spectra, the script
works with data acquired using two distinct types of acquisition methods: either a multiple period
/ multiple experiment method containing any number of product experiments or an IDA
(Information Dependent Acquisition) method. In the former case, the script calculates one
spectrum for each experiment by averaging all spectra acquired for the experiment. In the latter
case, the script uses all dependent product spectra, combining adjacent spectra with the same
parent mass and charge state.
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• If the active pane contains MS/MS data and the associated data file contains
more than one sample, click All MS-MS spectra from all samples for
current file to perform a single search using all MS/MS spectra from all
samples in the data file.
3. To open the Mascot search form and populate it with the appropriate information,
click Search. If you are searching all product spectra contained in the sample, this
may take some time (a progress bar will appear). After the Web form shows, click
Start Search.
4. To set the various search options, click Options.
Figure 1-22 Mascot Search-Options Dialog
Tip! To display the Mascot search form defaults Web page, click Set
default parameters.
5. To set where the text file used as input to Mascot is located, click Set search file
location, select one of the following options and then click OK.
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• To place the file in the Windows temporary folder with a random but unique file
name, click In the Windows ‘Temp’ folder with a random filename.
• To always write to a specific file, overwriting the file for every search, click
Always to the following file. To navigate to the folder, click Set.
• To create the file with a random but unique filename in the specified directory,
click To the following directory with a random filename. To navigate to the
folder, click Set.
• To be prompted to save every time a search is performed, click Prompt me
each time for the location.
6. In the Default precursor charge states group when searching only the current
spectrum, values here are not available; the precursor charge state should be set
manually using the resulting Mascot web form. When searching product spectra for
an IDA run, the charge state is automatically determined by the script—the values
specified here are not used unless the charge state could not be automatically
determined or the Try to determine charge state from survey scan check box is
not selected. When searching product spectra for a multiple period or multiple
experiment LC/MS run, the specified charge states will always be considered for
each product spectrum.
This option is only required for those using an older Mascot software version that
does not accept charge states greater than 5. If you are using one of these older
versions, make sure that in the Default precursor charge states group, the Discard
ions with charge of 5+ or higher check box is selected. Some versions of the Mascot
search engine cannot accept ions with a charge state of 5 or higher and show a
warning for each ion exceeding this limit.
7. To use the default precursor charge states as is, clear the Try to determine charge
state from survey scan check box. Otherwise, it will attempt to determine the
charge state by examining the isotope spacing in the survey spectrum, and for
Analyst TF 1.5 software data, it will use the charge state determined by the MS
Acquisition Engine, which is saved to the data file. If this check box is selected but
the charge state determination fails, the default charge states are used.
8. In the MS/MS averaging of IDA dependents group, edit the parameters that pertain
to the calculation of the product ion spectra for an IDA run.
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Note: If you have used the dynamic exclusion IDA option, set this value to
1.
9. In the MS/MS data processing group, select parameters that pertain to the filtering
of product ion spectra.
• Remove peaks if intensity < _ — Removes peaks that are either less than a
specific count or a specific percent of the maximum peak intensity of the
spectrum.
• To centroid the MS/MS spectra before sending them to Mascot for searching,
select the Centroid all MS/MS data check box. It is highly recommended that
you enable the centroid option.
• If the centroid option is used, indicate whether isotope peaks should be
removed from the MS/MS spectra before sending them to Mascot by selecting
the De-isotope MS/MS data check box. It is recommended that you enable
this option.
• Report peak area (otherwise intensity)—If selected, the script uses the area
of the peak. Otherwise it uses the intensity at the apex.
• Reject spectra if less than ‘n’ peaks —If a spectrum contains unreasonably
few peaks after combining adjacent spectra (if used) and centroiding (if used),
the spectrum can be eliminated.
• Remove peaks within ‘n’ Da of precursor m/z— Sets a window around the
precursor ion m/z and then removes any peaks within that window.
10. In the Other group, select Use original format for query titles if you are using a
third-party protein quantitation application and you would like to use the original title
format.
11. To display the Mascot search form defaults Web page, click Set default
parameters. You can edit the various defaults so that you do not need to reset them
manually every time before submitting a search. After changing the parameters, click
Save defaults as cookie to close the Web page.
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2. In the Parent Formula field, type the formula for the parent ion. The Mass Defect
and Nominal Mass fields are automatically updated.
3. If the parent formula is unknown, then type values in the Nominal Mass and Mass
Defect fields.
4. In the Mass Defect Tolerance (+-) field, type the tolerance value.
5. If required, type a value in the Resolution Factor field. The Resolution Factor
further filters the data by keeping only the centroid values whose resolution is
greater than or equal to it.
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6. If required, to allow the mass defect to be applied differently at each nominal mass in
the spectrum, click the Use Dynamic Mass Defect Calculation check box. If you
deselect the check box, a constant value of the mass defect is added to each
nominal mass.
7. In the Mass Range Parameters group, select the Use Mass Range Filter check
box to set the mass range parameters. Only masses in the spectrum between Start
Mass and Stop Mass inclusively will be retained.
8. Click OK to start processing.
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Merge MRM
Use this script to add all experiment data from the method to be merged to the base method.
Both methods should have only one period and one experiment. The script does not limit the
number of mass ranges in an experiment. All mass ranges are saved to the base method.
This script can be used to merge multiple final methods created by Compound Optimization.
After you have merged the methods, the LC information can be modified if necessary to reflect
the analysis conditions.
2. To navigate to the original acquisition method file or template, click the button to the
right of Original method.
3. To specify the name and location of the merged acquisition method file, click the
button to the right of New Method, type the name of the method, and then click
Open.
4. To add an acquisition method to the list of methods to be merged, click Add to the
right of Methods to be merged. To remove a method from the list, click Remove.
5. (Optional) If both methods were created using Compound Optimization, select the
Update MRM compound ID from the file name check box to populate the
compound ID column with the compound name in the merged method.
6. To add all the mass ranges from the selected methods, click Go.
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Item Description
1 Status Window
2 Log File
3 Main Controls
4 Overall Progress
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4. Enter the compound information required for the optimization process and then click
OK on the Settings dialog.
5. To initiate the optimization process, click Start in the MRM3 Optimization window.
Calculate m/z
The m/z calculator is accessed through the Settings dialog.
1. In the MRM3 Optimization window, click Settings.
2. In the Settings dialog, click Calculate from chemical formula.
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3. In the Chemical Formula field, type the chemical formula of the compound. Use
capital letters for elements. The chemical formula for peptides is also entered into
this dialog. You can obtain the chemical formulas for peptides by typing the peptide
sequence into the New Protein Sequence window in the BioAnalyst™ Software.
4. In the Num of charges field, click the number of charges.
5. To calculate the m/z for the entered chemical formula and charge, click Calculate.
6. To close the calculator and update the Expected m/z (amu) field in the Settings
dialog with the calculated m/z, click Use m/z.
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3. In the Scan Rate fields in the Enhanced Resolution, Enhanced product Ion and MS/
MS/MS groups, select a scan rate for ER, EPI, and MS3.
4. In the Q1 Multiple Ion group, in the DP Ramp fields, type the declustering potential
(DP) range for optimization. The range is expressed in absolute values and the
appropriate polarity is automatically applied based on the selection made in the
Settings dialog.
5. In the Enhanced Product Ion group, do the following:
• In the 2nd Precursors field, type the maximum number of second precursors
(fragment ions) used for MS3 optimization. Type a number between 1 and 10.
• In the Enhanced Product Ion group, in the Mass range field, type a mass
range for the second precursors that will be selected for MS3 optimization.
• In the CE field, type a collision energy value and in the CES field, type a
collision energy spread (CES) value that will provide a good MS/MS spectrum
from which fragment ions can be selected.
6. To generate all of the final MS3 methods for each second precursor and the optimal
MS3 method for quantitation, in the Generate Final Methods group, click Save All
Final Methods. Click Save Optimal Method Only to save only the optimal MS3
method (most sensitive for quantitation).
7. Click OK to accept the updated Advanced Settings.
Optimization in Progress
When the optimization is started, Manual Tune in the Analyst® software is automatically stopped.
While the script is running, all of the functions in the software can still be used. A Log.txt file is
also updated as each part of the optimization procedure is completed. To stop the script at any
time, click the Abort button. Examples of the script in progress are shown in the Figure 1-30 on
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page 60 and the Figure 1-31 on page 61. In the Overall Progress section, the Checklist images
and text fonts represent different statuses that are described in the following section.
When the text is underlined, you can click it like a web page hyperlink and the corresponding
spectrum or chromatogram is displayed. The text found under MS/MS/MS also displays the MS3
scan number that is being performed because it is possible to have between 1 and 10 scans. The
Overall Progress section also includes a Message area. In this area, a progress bar displays the
current step progress. Above the progress bar, various messages are displayed such as the time
and other statuses for the current optimization step.
Figure 1-30 MRM3 Optimization Window after EPI Scan
Item Description
1 Checklist
2 Message
In the spectral status window, the previously generated spectrum or chromatogram is displayed.
When one of the checklist items is selected, the corresponding graph is displayed. The scan type
name indicates which scan is currently being displayed. For each completed step, it is possible to
open the acquisition method (.dam) or data file (.wiff) associated with the graph displayed. If an
MS/MS/MS scan is displayed, you can use the buttons to cycle through the different MS3 scans.
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1 2 3
Item Description
1 Scan type
2 Buttons to cycle through different MS3 scans
3 Links
Optimization Complete
When the quantitative optimization for MS3 is completed or stopped, a Results.txt file is
generated. This file is automatically opened in Microsoft Notepad. You can also view the file by
clicking View Results from the MRM3 Optimization window. The various parts of the Results.txt
file are described as follows.
• Time and Duration: Displays the date and time duration of optimization.
• User Starting Conditions: Displays the settings and Advanced Settings in this
section.
• Optimization Conditions Found: Displays the optimal conditions found during the
ER and Q1MI scans.
• MS3 Fragments Found and Associated Losses: Displays the fragments and
optimal conditions (collision energy and excitation energy) as well as associated
losses found for the EPI scan and MS3.
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3 4
Item Description
1 Time and Duration
2 User Starting Conditions
3 Optimization Conditions Found
4 MS3 Fragments Found and Associated Losses
All of the generated acquisition methods have a descriptive file name in the format [supplied
compound name] + [scan type] + [m/z] + .dam. These methods are saved in the same folder as
the starter acquisition method.
All of the data, Log.txt, and Results.txt files are saved into a Data sub-folder that is created in the
same project as the starter acquisition method. The sub-folder has the format [supplied
compound name] + OptMS3 + ([date], [time]). The data files have the format [supplied compound
name] + [scan type] + [m/z] + .wiff.
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Initialization
Before performing any optimization scans, the MRM3 Optimization script first performs the
following initialization steps. If an error occurs during any of these steps, the script will stop the
optimization process.
1. Ensure that the Analyst software is running.
2. Load the starter acquisition method to see if it is valid and check the device type.
3. Create a new Data sub-folder to store the .wiff files.
4. Create the Log.txt file.
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with 2 V step and Dwell Time of 50 ms. Each overlaid graph is then smoothed two times and the
voltages yielding the greatest ion count are used as the optimal CE values.
MS/MS/MS Scan
The script performs an MS3 scan for each chosen second precursor at the specified scan rate
and with an AF2 ramp of 0 to 100 V with 2 mV step (or 0 to 0.4 V with 0.01 V step on QTRAP®
5500 system) for both polarities. The fill time of the scan is set, and Q0Trapping can be turned on
for maximum sensitivity if required. The lower limit of the mass range for the MS/MS/MS scan
can be specified, and the upper limit is second precursor + 5 amu.
The generated graphs are smoothed twice and the optimal AF2, as shown in the Figure 1-33
How AF2 is Determined, is obtained when the residual intensity of the second precursor (based
on XIC) is at 5% of its maximum intensity. The spectrum at this AF2 value is then used to find the
two most intense second generation fragment ions, excluding peaks within ±1 amu of the second
precursor. If the second precursor m/z is greater than 10% of the total ion count, no fragments
from that spectrum will be used. This condition exists because if the second precursor m/z is
greater than 10%, there is insufficient fragmentation.
Figure 1-33 How AF2 is Determined
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2. To attach an additional script to this batch, click Add Script to navigate to the
acquisition script.
3. To remove a script, click the script and then click Remove Selected.
4. Select the Only show this dialog again if the control key is down check box if
you want the dialog to appear when submitting the batch.
5. Click Run to attach all of the acquisition scripts to the batch.
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Open in Workspace
Use this script to open a sample in a previously created workspace. This script loads the
previous workspace and displays the sample in the same layout as specified in the saved
workspace. It is not possible to create a workspace that specifies pane arrangements so that any
sample can be loaded into it. For more information on workspaces, refer to the online Help.
2. To select a data file from another project, click File > Open Data File.
3. In the Data files list, click a data file.
All the samples in the selected data file appear in the Available Samples list
4. After choosing the sample, select a workspace in the Available workspaces list. To
select a different workspace, click Workspace > Open Workspace.
5. To set a default workspace to be used each time this script is run, select the Set
Selected Workspace As Default check box.
6. To open the sample, click Open Sample in Workspace.
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Regression Calculator
Use this script to calculate the slope, y intercept, and r values for each mass/speed dependent
parameter.
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Savitzky-Golay Smooth
Using the Analyst® software, you can smooth a graph in Explore mode using two different
algorithms. When you are using the IntelliQuan integration algorithm in Quantitate mode, a third
smoothing algorithm, the Savitzky-Golay smooth, is available. The active graph in the Analyst
software will be replaced by the smoothed graph.
The Savitzky-Golay Smooth script smooths the current active graph using the Savitzky-Golay
smoothing algorithm. The IntelliQuan algorithm in Quantitate mode performs the smoothing
process.
2. Use the Smoothing Half Width drop-down list to set the half-width for smoothing
the data. The total width will be twice this value plus one. This parameter is the same
as the smoothing parameter used with the IntelliQuan algorithm in the Analyst
software.
3. To perform the smoothing, click OK.
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Calculate the Average and Standard Deviation of a Graph More than Once
1. Copy the graph into a new pane. Click Explore > Duplicate Data > Same Window.
2. Make a selection in the new graph pane and run the Selection Average and
StdDev script again.
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Prerequisites
You must have the SpecManager software installed.
1. With either a chromatogram or a spectrum in an active pane, click Script > Send to
ACD SpecManager.
The following figure shows the options dialog that opens when you run either of the
scripts to process chromatographic data. If you are interactively processing a single
spectrum active in the Analyst® software, these options do not apply.
2. To centroid the exported spectra, select the Centroid Exported Spectra check box.
This option reduces the size of the exported JCamp file.
3. To select the threshold that will be applied to the exported spectra, in the Threshold
field, type a value. If you do not want to use a threshold, type 0 in the Intensity
Threshold field.
4. Select the Only show this dialog again if the control key is down check box to
have the JCAMP Options dialog appear if the Ctrl is pressed when selecting the
script from the Script menu or when submitting the batch to the queue.
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5. Click OK to continue processing and to have the spectra exported. These values are
used as defaults until you change them again.
6. To close the dialog without making any changes, click Cancel. In the case of
interactive use, canceling the dialog will also stop the export operation. However, in
the case of batch operation, the batch will still be acquired and JCamp files will be
exported using the original parameters.
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Tip! To remove the labels, press the Ctrl key while clicking the script.
Related Scripts
S_NstdDevQS: Calculates the signal-to-noise value with a method that uses the noise regions
standard deviation.
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2. To erase any labels current on the active chromatogram, click Erase Labels.
3. To calculate the signal-to-noise ratio and label the graph, click Go.
Related Scripts
S-to-N using Peak-to-PeakS: Calculates the signal-to-noise for an active chromatogram. The
background subtracted signal is divided by the peak-to-peak noise level.
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Note: Make sure that the mass (or time) range for splitting is displayed. If
necessary, dock the graph to make sure that the whole graph can be seen.
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Unit Conversion
Use this processing script to convert from one set of concentration units to another.
2. In the Convert from field, type the concentration and units to be converted.
3. If the conversion is from a weight-based concentration (for example, g/L) to a molar-
based concentration (for example, mol/L), then in the MW field, type the molecular
weight of the component.
4. In the Convert to field, type the unit.
5. To perform the conversion, click Convert.
The calculated values will be displayed in the Convert to field.
6. To retrieve these values, press Ctrl +C to select and copy them to the clipboard.
These values can then be pasted into another application.
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Wiff to MatLab
Use this script to extract the data from a .wiff file and creates the following matrices in a MatLab
file: Data, Masses, Wavelengths, Scans, and Filename. For more information about these
matrices, refer to Table 1-8 Matrix Definitions on page 83. This MatLab file can then be included
in a MatLab script and used to compute and display the results.
Although you can use the Analyst® software to perform various data manipulations, you may use
this specialized script, Wiff to MatLab, for data computations. The Analyst software cannot
extract data from a .wiff file and then store it in a file that can be read and interpreted by MatLab.
In addition to exporting the mass spectral data, this script can also export data from a diode array
detector (DAD).
The script will create a single MatLab .mat file for each sample to be translated. Depending on
the type of mass spectra data and the user options, the script can create data in two different
formats:
• The first format saves the intensities in a matrix of size (number of masses) x
(number of scans). There is thus an entry in the matrix for every mass that was
scanned and every spectrum. This is the default format for quadrupole spectra and
the format that is always used for diode array data.
• The second format is sparse or compressed and allows data points that have an
intensity of zero to be omitted; depending on the number of such data points in the
original .wiff sample, this can potentially greatly reduce the size of the MatLab file.
This format uses a matrix of size 2 x (total number of (mass, intensity) pairs). The
first row represents masses and the second row the intensities. A given number of
initial columns corresponds to the first spectrum, a given number of following ones to
the second spectrum, and so forth. Each (sparse) spectrum is essentially stored
end-to-end.
A separate vector is written, which you can use to determine the starting and ending position of
any given spectrum in this matrix. This vector contains the one-based index of the start of a given
spectrum. The end for a given spectrum can be determined by subtracting one from the start of
the following spectrum (except for the very last spectrum, which is determined by the size of the
above-mentioned matrix).
The first format is the default format for quadrupole spectra and is always used for diode array
data. The second (sparse) format is always used for TOF (time-of-flight) data and can optionally
be used for quadrupole data.
The actual names of the various matrices are specified using the Options dialog described later
in this document.
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3. In the Matrix Names fields, type the names of the matrices that the script produces.
It is recommended that you keep these names as their default values.
4. In the Threshold for sparse format field, type a value that will be used to reduce
the size of the output MatLab file. For the sparse format only, only (mass, intensity)
pairs with intensity larger than the specified value will be written.
5. If the Save original filename as matrix field is selected, then the script will create
and populate the Filename matrix.
6. To have the quadrupole spectra saved in the sparse format, select the Use sparse
format for quadrupole data check box. Note that TOF spectra are always saved in
the sparse format and that diode array data is always saved in the non-sparse
format.
7. If the Save scan times (not scan numbers) field is cleared, then the script will
populate the Scans matrix with the time in seconds for each scan. Otherwise, the
Scans matrix is populated with the scan numbers.
8. In the Use fixed step size field, type the step size of the data to extract. If this field is
cleared, then the acquisition step size is used.
9. To populate the wavelengths matrix with the DAD data when the script is attached to
a batch, select Save DAD data in batch mode (if available).
10. To save these settings and continue processing the data, click Save.
11. To discard any changes made to the settings, click Don’t Save. Data processing will
continue after you click this button.
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12. To automatically generate a MatLab file for every sample submitted as part of a
batch run in the Batch Editor, click Select Script to select the script before
submitting the batch to the queue. The script should be located in the Processing
Scripts subproject of the API Instrument project.
A MatLab file is created for every different experiment for each sample in the batch.
The MatLab files are placed in the same location as the data files and will have the
same names with the .wiff extension replaced by .mat; however, the index of the
sample within the .wiff file will be appended. In addition, for samples acquired using
acquisition methods containing more than one experiment, the period and
experiment number will be appended to the MatLab filename.
For example if a data file is called test.wiff and contains two samples, the MatLab
files will be called:
• test-1.mat
• test-2.mat
If the acquisition method contained one period and two experiments, four MatLab
files would be generated (two for each sample):
• test-1(1,1).mat
• test-1(1,2).mat
• test-2(1,1).mat
• test-2(1,2).mat.
If the data file contains diode array data and the Save DAD data check box is
selected, then an additional file with the sample index and (DAD) is created. For the
previous example, files called test-1(DAD) and test-2(DAD).mat are created.
Related Scripts
Export to JCamp: Converts spectra from .wiff format to JCamp format.
Table 1-8 Matrix Definitions
Matrix Dimensions Type Descriptions
Data Number of Float The raw intensities for all of the spectra.
masses (or
wavelengths) x
number of scans
SparseData 2 x total number Double The raw masses (first row) and intensities
of (mass, (second row) for all spectra. This matrix is
intensity) pairs used only with the sparse format.
Masses Number of Float The actual m/z values scanned by the
masses x 1 instrument. This matrix is only present when
exporting MS data.
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When processing is started, a progress bar indicates the current step. When
finished, a new pane with overlaid XIC traces is the active pane in the Analyst®
software document. If you want to use the Cycle Overlays feature and it is
unavailable, switch the active pane in the Analyst software to another one and then
reselect the overlaid XIC pane.
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2. Using the grid in the dialog, type the mass ranges to be extracted into an XIC. To
populate the grid from a text file, click File > Open and then navigate to the text file.
3. To save the current information in the grid to a file, click File > Save As.
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