Deconvolution Plugin Software Manual

Deconvolution Plugin for
ISQ LT, TSQ 8000 Evo, and

Q Exactive GC
Mass Spectrometers
Software Manual
1R120706-0006 Revision B February 2016

© 2016 Thermo Fisher Scientific Inc. All rights reserved.
Thermo Fisher Scientific Inc. All rights reserved.

Exactive, Orbitrap, TraceFinder, and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc. in the
United States.
The following are registered trademarks in the United States and possibly other countries: Adobe and Reader
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registered trademarks of Microsoft Corporation.
Data Dependent and Q Exactive are trademarks of Thermo Fisher Scientific Inc.
All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use in the
product operation. This document is copyright protected and any reproduction of the whole or any part of this
document is strictly prohibited, except with the written authorization of Thermo Fisher Scientific Inc.
The contents of this document are subject to change without notice. All technical information in this
document is for reference purposes only. System configurations and specifications in this document supersede
all previous information received by the purchaser.
This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a purchaser. This
document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of
Sale shall govern all conflicting information between the two documents.
Release history: Revision A released in July 2015; Revision B released in February 2016.
Software version: Exactive Series 2.7 or later; TraceFinder 4.0 or later
For Research Use Only. Not for use in diagnostic procedures.

C
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .v
Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Cautions and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .viii
Deconvolution Plugin Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Setting Up the Deconvolution Plugin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Functional Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Sample View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Acquiring Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Processing the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Filtering Processed Data and Removing Unwanted Peaks . . . . . . . . . . . . . . . 18
Cross-Sample Compound Alignment (Optional) . . . . . . . . . . . . . . . . . . . . . 22
Exporting Library Hits to the NIST Library . . . . . . . . . . . . . . . . . . . . . . . . . 25
Importing Results into TraceFinder Software . . . . . . . . . . . . . . . . . . . . . . . . 28
Creating a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Thermo Scientific Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) iii
P
Preface
This guide provides reference information about the parameters in the Thermo Scientific
Deconvolution Plugin application within Thermo Scientific™ TraceFinder™ software for gas
chromatography (GC) and mass spectrometry (MS) instruments. The Deconvolution Plugin
can be used with the following GC-MS systems:
• Thermo Scientific™ Q Exactive™ GC Mass Spectrometer
• Thermo Scientific™ ISQ™ LT Mass Spectrometer
• Thermo Scientific™ TSQ™ 8000 Evo Mass Spectrometer
Contents
• Related Documentation
• System Requirements
• Cautions and Special Notices
• Contacting Us
Related Documentation
The Deconvolution Plugin application includes complete documentation. In addition to this
guide, you can also access the following documents as PDF files from the data system
computer:
If you have a Q Exactive GC mass spectrometer, the manuals on your data system are:
• Q Exactive GC Preinstallation Requirements Guide: PN 1R120706-0001
• Q Exactive GC Operating Manual: PN 1R120706-0002
• Q Exactive GC Software Manual: PN 1R120706-0003
• Q Exactive GC Quick Start Guide: PN 1R120706-0004
 To view the product manuals
Go to Start > Programs > Thermo Exactive Series > Manuals.
Thermo Scientific Deconvolution Plug In Software Manual (P/N 1R120706-0006, Revsion B) v

Preface
Related Documentation
If you have a TSQ 8000 Evo mass spectrometer, the manuals on your data system are:
• TSQ 8000 Evo Preinstallation Requirements Guide: PN 1R120568-000
• TSQ 8000 Evo User Guide: PN 1R120568-0002
• TSQ 8000 Evo Hardware Manual: PN 1R120568-0003
• TSQ 8000 Evo Spare Parts Guide: PN 1R120568-0004
• TSQ 8000 Evo AutoSRM User Guide: PN 1R120568-0005
Open the Manuals folder on your desktop.
If you have an ISQ LT mass spectrometer, the manuals on your data system are:
• ISQ Series Preinstallation Requirements Guide: PN 1R120555-0001
• ISQ Series Hardware Manual: PN 1R120555-0002
• ISQ Series User Guide: PN 1R120555-0003
• ISQ Series Spare Parts Guide: PN!r120555-0004
Open the Manuals folder on your desktop.
 To open Help
• From the Deconvolution window with the TraceFinder software application, choose
Help.
For access to the application Help, follow this procedure.
 To view application-specific Help
• From the application window, choose Help > Deconvolution Plugin Help.
• If information about setting parameters is available for a specific view, page, or dialog
box, click Help or press the F1 key for information about setting parameters.
• In applications that have a Communicator bar, click the field or parameter to display
definitions, required actions, ranges, defaults, and warnings.
For more information, visit www.thermoscientific.com.
vi Deconvolution Plug In Software Manual (P/N 1R120706-0006, Revsion B) Thermo Scientific

Preface
System Requirements
System Requirements
The Deconvolution Plugin application requires a license. In addition, ensure that the system
meets these minimum requirements.
IMPORTANT Before you install the device driver, ensure that the data system computer
has a compatible version of the Thermo Foundation™ platform and instrument control
software as noted in the Deconvolution Plugin 1.1 Release Notes.
System Minimum requirements

Computer • 4.6 GHz processor with 16 GB RAM
• CD/R-Rom or DVD drive
• 1000 GB or hard drive
• Video card and monitor capable of 1680  1050 resolution
• Quad core processor
Software • Adobe™ Reader™ 10
• Microsoft™ Windows™ 7 SP1 (64-bit)
• Thermo Foundation™ 3.1 SP1
• Thermo Scientific™ Xcalibur™ 4.0
• TraceFinder 4.0
• Instrument Control Softwarea
– GC Devices 3.2 or later
– Exactive Series 2.7 or later (Q Exactive GC systems)
– TSQ Series 3.2 or later (TSQ 8000 Evo systems)
– ISQ Series 3.2 or later (ISQ LT systems)
a
Check latest release notes for version compatibility.
Cautions and Special Notices

Make sure you follow the cautions and special notices presented in this guide. Cautions and
special notices appear in boxes; those concerning safety or possible system damage also have
corresponding caution symbols.
This guide uses the following types of cautions and special notices.
Thermo Scientific Deconvolution Plug In Software Manual (P/N 1R120706-0006, Revsion B) vii
Preface
Contacting Us
CAUTION Highlights hazards to humans, property, or the environment. Each CAUTION

notice is accompanied by an appropriate CAUTION symbol.
IMPORTANT Highlights information necessary to prevent damage to software, loss of

data, or invalid test results; or might contain information that is critical for optimal
performance of the system.
Note Highlights information of general interest.
Tip Highlights helpful information that can make a task easier.
Contacting Us
There are several ways to contact Thermo Fisher Scientific for the information you need.
 To find out more about our products
Go to www.thermoscientific.com/en/products/mass-spectrometry.html for information

about our products.
 To get local contact information for sales or service
Go to www.thermoscientific.com/en/support-landing/support.html.
 To suggest changes to documentation or to Help
• Fill out a reader survey online at www.surveymonkey.com/s/PQM6P62 or send an

e-mail message to the Technical Publications Editor at
techpubs-austin@thermofisher.com.
viii Deconvolution Plug In Software Manual (P/N 1R120706-0006, Revsion B) Thermo Scientific
Deconvolution Plugin Application
This chapter contains set-up information about, detailed descriptions of the functions, and a
sample workflow for the Deconvolution Plugin application for TraceFinder software.
Contents
• Setting Up the Deconvolution Plugin
• Functional Description
• Workflow
Setting Up the Deconvolution Plugin

 To set up the Deconvolution Plugin
1. First, ensure the TraceFinder 4.0 software and the Deconvolution Plugin application are
installed. If not, follow the instructions in the release notes or installation help files to
install the programs.
2. Click the TraceFinder 4.0 Administration Console icon on your desktop. The
Administrator Console opens.
3. In the Administrator Console, double-click Administration under Plugins. See Figure 1.
Note If you have enabled security, an administrator must log in to the TraceFinder
Administrator Console before modifying plugin settings.
Thermo Scientific Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) 1

Setting Up the Deconvolution Plugin
Figure 1. Configuring the Plugin Using the TraceFinder 4.0 Administrator Console
4. Check the box labeled Thermo.Deconvolution and click Apply to enable the
Deconvolution Plugin application. See Figure 2.
Figure 2. Enabling the Deconvolution Plugin
5. Restart the TraceFinder application to view the plugin.
2 Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) Thermo Scientific

Functional Description
IMPORTANT If you want to use TraceFinder software itself to process data by unknown
screening, you must uninstall the Deconvolution plug or your analysis will return no
results. To uninstall the plugin, deselect Thermo.Deconvolution in the TraceFinder
Administrator Console and restart the TraceFinder application.
This section contains descriptions of the functions of the Deconvolution Plugin application
for TraceFinder software.
Sample View
The sample view of the Deconvolution Plugin application contains the following sections:
• Sample List
• Peak List
• Peak Identification
• Total Ion Chromatogram
• Extracted Ions
• Spectra
Figure 3 shows the Sample View of the Deconvolution Plugin application.

Figure 3. Deconvolution Plugin Sample View

Sample List
The Sample List window displays the names and identifying information of the samples in the
batch. To switch between samples in a batch, click on the sample in the table. See Figure 4.
Figure 4. Sample List Window
Peak List
The Peak List window allows users to switch between chromatographic peaks which have
been detected. The peak sets are named by the compound selected by the user in the Matched
Compounds in the Peak Identification list. The peak list also lists retention times and
summed ion intensities. See Figure 5.
Figure 5. Peak List Window
Peak Identification
Figure 6. Peak Identification Window
Export to
NIST Icon
The Peak Identification window uses the following criteria to identify compounds.

• Matched Compound — The primary name of a compound in the selected library.

• Library Hit Name — Alternate names for a compound that appear in the selected
library.
• Formula — The compound formula for each detected peak.
• CAS Number — The CAS number for each compound detected.
• SI — The search index score (0-999) for each compound detected returned by the NIST
library search.
• HRF (High-Resolution Filtering) Score — The percentage of the total ion
chromatogram of the spectrum that can be explained by the chemical formula in the
library search. A complete set of theoretical ions which could result from the chemical
formula is generated from all non-repeating combinations of atoms. A simple example is
the chemical formula C2H2, which could produce the ions H+, H2+, C+, CH+, CH2+,
C2+, C2H+, and C2H2+. Each peak in the deconvolved experimental spectrum is
checked to see if it is within the set mass tolerance of one of these possible ions from the
original chemical formula. The intensity of the ions which match at least one possible ion
from the original chemical formula is divided by the total ion current of the deconvolved
experimental spectrum to give the HRF Score.
• M+ m/z —The mass-to-charge of the molecular ion.
• Score — Combined score (0-100) indicating quality of match between this library hit
and the deconvolved experimental spectrum. It combines several metrics including SI and
HRF Score.
• M+ — This value is set to Yes if the molecular ion is detected in the deconvolved
experimental spectrum. See the Extracted Ion View.
• M+ Lib — The value is set to Yes if the NIST spectrum has an ion at a m/z consistent
with the molecular (M+) ion.
• % Elements — This value is the percent of all the elements in the library hit formula that
are found in at least one theoretical ion from the chemical formula assigned to a m/z in
the deconvolved experimental spectrum.
• Library — The name of the database used for the compound identification.
You can also switch between compound matches for a selected chromatographic peak set and
select the correct compound ID. If you want to select a different compound than the one
selected, right-click and choose Select Library Hit. If you want to deselect a
compound—normally done if none of the listed compounds is the correct
identification—highlight it in the list, right-click and choose Deselect Library Hit. Peaks
without a selected library hit are named “Peak@x.xxx min.” Library searches can be done on
all or selected compounds. Users can export the accurate-mass deconvoluted spectra from the
acquired compound to a .msp file that can be imported into the NIST MS Search program by
clicking the Export to NIST Library Icon. See Figure 6. For instructions on exporting
deconvolved spectra to the NIST Library, go to Exporting Library Hits to the NIST Library.

Total Ion Chromatogram

The Total Ion Chromatogram window displays a plot of the total ion chromatogram (TIC).
Users may zoom in and out on chromatographic peaks. To view the TIC, select View >Total
Ion Chromatogram from the top menu of the Deconvolution application. The selected
chromatographic peak is highlighted. Users can select the active peak set by clicking on the
marks in the TIC. The extracted ions for the selected binned peak set should be visualized at
its retention time. See Figure 7.
Figure 7. Total Ion Chromatogram Window
Extracted Ions
The Extracted Ions window displays an overlay of the extracted ion chromatogram peak set. It
contains a list of ions found in the deconvolved experimental spectrum.The selected mass is
always highlighted, and users can select different masses using this window. To select
individual masses, click on the ion trace of interest in the window or click one of the rows in
the table. See Figure 8.
The selected ion is also highlighted in the Spectra window. See Figure 9.
Figure 8. Extracted Ions Window
Active Ion Trace

(in Red)
Reprocess Library
Search Icon
Active Ion Trace

(Selected in Table)
The mass list for the selected peak is displayed underneath the Extracted Ions window. The
mass list has the following elements:

• Active — Lists whether or not the mass is active in the spectrum. All masses in the
spectrum are defaulted to active.
Note Users can deactivate ions by removing the check from the Active box. Reprocessing
the library search removes inactive ions from the spectrum.
• Measured m/z — The measured mass-to-charge ratio of the active mass.

• Area — This refers to the area under a peak for the measured m/z.
• Height — This refers to the peak height for the measured m/z.
• Fragment ID — The predicted fragment formula with the smallest mass error. If there is
more than one fragment that could match the measured m/z, right-click and choose Show
All Fragments For Mass to view the list of all possible fragments.
Note If fragment annotation was not selected in the processing parameter window when
the batch was processed, right click within the mass list table and select Annotate
Spectrum to identify the ions within the selected compound's spectrum.
• Fragment Count — This parameter refers to the number of theoretical fragments of the
library search formula that fall within the set mass tolerance of the experimentally
measured m/z. If this value is ≥1, we say this m/z is “explained” by the formula from the
library hit. All ions with ≥1 fragment count are used to calculate the HRF Score.
Note To view all fragments for ions with ≥1 fragment count, right-click on the ion in the
Fragment Count list and select Show Other Fragments for Mass.
• M+ — (To view this parameter, click the magnifying glass in the Extracted Ions top
menu) This value is set to True if this ion is within the set mass tolerance of the
theoretical M+ of the chemical formula returned by the library search. This value is also
set to True if no theoretical ion matches this ion. The M+ Yes/No in the Peak
Identification View is not set to Yes unless an M+ was actually found in the spectrum.
• Theoretical m/z — The mass-to-charge ratio as calculated from each Fragment ID.
• Mass Error (ppm) — The relative mass error for the measured m/z relative to the
theoretical m/z.
Spectra
The Spectra window displays the acquired deconvoluted spectra with mass precision to six
decimal places for high-resolution date (two decimal places for unit-mass data) along with the
currently selected library spectrum for comparison. The selected mass is highlighted. Users
can select different masses in this window. Clicking on a mass peak shows the exact mass as
well as the formula for the closest matching fragment. See Figure 9.

Workflow
Figure 9. Spectra Window
Workflow
The information below provides a sample workflow for the Deconvolution Plugin software.
The workflow described here consists of the following sections:
• Acquiring Data
• Processing the Data
• Filtering Processed Data and Removing Unwanted Peaks
• Cross-Sample Compound Alignment (Optional)
• Exporting Library Hits to the NIST Library
• Importing Results into TraceFinder Software
• Creating a Report
For additional information, refer to the TraceFinder software user manuals or your mass
spectrometer’s user guide or software manual.
Acquiring Data
The first step in your workflow is to acquire data in the TraceFinder software application.
 To acquire data in TraceFinder software
1. First, go to Method Development in TraceFinder and create an unknown screening

master method. See the TraceFinder software user documentation for instructions.
2. Create a batch in the TraceFinder application. Select File > New > Batch. See Figure 10.
You may also use the acquisition wizard in TraceFinder to acquire your batch.
Note It is recommended that you deselect Alignment and Gap Filling when setting up
your TraceFinder software Master Method. This turns off the With RT Alignment
processing function when submitting a batch for acquisition in TraceFinder software.

Workflow
Figure 10. Creating a Batch
3. The Create New Batch window opens. Name the batch, set Type to Unknown Only and
select the master method you created in step 1 for your sequence. See Figure 11.
Note The Deconvolution Plugin application can only process full-scan MS scan filters,
and only one scan filter per raw file. Use the guidelines below to determine if the
application can process your raw file.
If you only want to peak detect and library search a raw file, and you have:
• A raw file with two full-scan MS filters that are identical except that one is a lock
mass, the application uses the scan filter without the lock mass to process the raw file.
• A raw file with more than one non-identical full-scan MS filter, the application does
not process the raw file and displays an error message.
• One full-scan MS filter (plus an optional matching lock mass filter) and a SIM, MSX,
PRM or other type of scan filter, the application uses only the full-scan MS filter
without the lock mass to process the raw file.
If you want to retention time align multiple files, all the raw files in your batch must meet
the above criteria and have identical full-scan MS filters.

Workflow
Figure 11. Naming a Batch
4. Set up your sample list and click the acquire sample icon to acquire your samples. See
Figure 12.
Figure 12. Acquiring Samples
Acquire Sample
Icon Group Names

Workflow
Note If you already have acquired raw files and want to associated them with this batch,
you may browse them in by double-clicking on a cell under Filename and choosing the
raw file or files.
Note Name groups in the sequence and the application will use those groups to calculate
fold changes and cv’s. If you name a group “control,” the software will use that as the basis
with which to calculate the fold change. Samples with the same group name will be placed
in the same group.
Processing the Data

Functional descriptions of the processing parameters and the workflow for processing data in
Deconvolution Plugin application can be found in the following sections:
• Functional Description of Processing Parameters
• Data Processing Workflow
Functional Description of Processing Parameters
 To access the processing parameters in the Deconvolution Plugin application
1. Click the processing icon in the Peak List window. See Figure 13.
Figure 13. Locating the Processing Icon in the Peak List Window
Processing Icon
2. The Processing Parameters window opens. See Figure 14.

Workflow
Figure 14. Viewing the Processing Parameters
3. Descriptions of available processing settings and how best to use them are below.
• Accurate Mass Tolerance (+-) — Enter the accurate mass tolerance that the software
uses throughout the program.
• m/z SigToNoise Threshold — Enter the signal-to-noise threshold that the software
uses to determine whether to include ions in each compound’s deconvolved spectra.
• TIC Intensity Threshold — Enter the TIC intensity threshold that the software uses
to determine whether to include compounds in the compound peak list. The TIC
intensity is the summed intensity of all ions in a compound’s spectra after passing
through the deconvolution processing step.

Workflow
• Ion Overlap Window — Enter the peak height at which the software creates a
retention time window around each compound’s base peak in order to determine
whether other closely eluting ions are grouped with this compound during the
deconvolution process. If a lower intensity ion falls within this retention time
window, and all other criteria is met (e.g. signal to noise threshold) it is grouped
together with the base peak and other ions meeting this criteria to create the
compound’s deconvolved spectra. The higher the ion overlap window percentage, the
less likely ions generated from two compounds are grouped within the same
spectrum.
• RT Aligning — Enter the retention time window that the software uses to look for a
compound detected in one sample in all other samples during the retention time
alignment process.
• Library Search Type — Select Normal or HiRes from the Library Search Type
drop-down menu. If the method uses an accurate mass library, select HiRes. If the
method uses a unit resolution library select Normal.
• Libraries — Choose the library or libraries you want to use, adjust the other settings
as desired, and click Save. The available libraries depend on those installed on your
system.
• SI (RSI) — Enter the search index threshold that the software uses to determine
whether to include a library hit in the Peak Identifications list. If a compound has
no library hits above the entered threshold, it is still displayed in the Peak List as an
unidentified peak. If it is desired to use the reverse search index rather than the search
index, select the Reverse Search box.
• Penalize Missing Molecular Ion — Check this box if it you wish to award extra
points to a library hit’s total score when the spectrum contains a molecular ion that
matches the library hit’s molecular formula within the set accurate mass tolerance. If
this check box is not selected, all library hits will receive the points awarded for
having the molecular ion of the library hit present in the spectrum.
• Annotate Fragments — Check this box if you wish to annotate fragments of each
identified compound during the library search processing step. This adds additional
processing time. Identified compounds can be annotated individually after processing
by right-clicking in the table at the bottom of the Extracted Ion Window (see
Figure 8) and selecting Annotate Spectrum.
Data Processing Workflow
 To process your data in the Deconvolution Plugin application
1. Click Open Deconvolution Plugin on the top menu of the TraceFinder application or
click the Deconvolution Plugin tab along the side menu. See Figure 15.

Workflow
Figure 15. Opening the Deconvolution Unknowns Plugin
2. (Optional) Click the pin icon on the Analysis tab to hide the side menu for better
viewing. See Figure 16.
3. Click the Refresh Sample List button to synchronize your sample list with the Batch
View. See Figure 16.

Workflow
Figure 16. Hiding the Side Menu and Refreshing the Sample List
Refresh Sample List
4. Click the settings icon to adjust the plugin settings. See Figure 17.
Figure 17. Locating the Settings Icon
Note For detailed descriptions of the processing settings, review the Functional
Description of Processing Parameters.

Workflow
5. The Settings dialog box opens. See Figure 18.

Figure 18. Changing the Settings
Note You must have NIST 2014 and at least one NIST-formatted library installed on
your system.
6. Select Normal or HiRes from the Library Search Type drop-down menu. If using an
accurate mass library, select HiRes. If using a unit resolution library select Normal. See
Figure 19.
Figure 19. Selecting the Library Type

Workflow
7. Choose the library or libraries you want to use, adjust the other settings as desired and
click Save. The available libraries depend on those installed on your system.
8. In the Peak List box, select whether you want the application to do peak detection or
peak detection and library search, and choose if you want to apply the settings to the
current sample, the unprocessed samples, or all the samples in your list. For this example,
we will select Peak Detection and Library Search applied to All Samples. See Figure 20.
Figure 20. Selecting the Peak Detection Settings
9. The plugin starts processing all the samples. The green status bars indicate the software is
processing your samples. This may take a significant amount of time if you have a large
number of samples or very large raw files. See Figure 21.
Note The Deconvolution Plugin application uses a lot of computer resources to process
data as quickly as possible. Processing results during acquisition can interfere with raw file
acquisition. Wait to process raw files until acquisition is complete or use separate
computers to acquire and to process data. Closing all other applications on the PC where
you are processing data will improve processing speed.

Workflow
Figure 21. Processing the Samples
Note You cannot use TraceFinder software while processing data in the Deconvolution
Plugin application.
Filtering Processed Data and Removing Unwanted Peaks

This section describes how to apply filters to processed data and remove unwanted spectra
from processed data. The workflow contains the following two subsections.
• Filtering Processed Data
• Removing Unwanted Peaks
Filtering Processed Data

This section describes how to apply filters to processed data.
 To set a compound filter
1. Click the compound filter icon. See Figure 22.

Workflow
Figure 22. Setting a Compound Filter

Compound
Filter Icon
2. The compound filter allows you to display only those compounds with a feature that you
are interested in. For example, as shown in Figure 23 below, the peak list can be filtered to
show only compounds with a deconvoluted spectra that contains a chlorine ion or
common chlorine fragments. Similarly, chlorine containing compounds can be filtered by
using the delta mass filter to pull out spectra that contain two mass differing by the mass
difference between Cl37 and Cl35 isotopes.
Figure 23. Filtering Chlorine Compounds
3. When this filter is turned on, the number of peaks is reduced from 235 to 29. See
Figure 24.

Workflow
Figure 24. Peak Number Reduced after Filtering
The following describes the various types of filtering that can be applied to a data set:
• Min TIC Intensity — Enter the TIC intensity threshold the software will use to
filter compounds in the compound peak list.
• Use RT Range — Enter the minimum and/or maximum retention time the software
will display compounds in the compound peak list.
• Use Mass Filter — Select if filtering by a mass or mass delta is desired.
– Spectra Contains Mass — Select to filter peak list to only display peaks that
contain a certain mass or masses. If any of the masses in the mass filter are
contained within a compound’s spectrum, the compound will be displayed.
– Spectra Contains Mass Delta — Select to filter peak list to only display peaks
that contain a certain delta mass or delta masses. If any two ions within a
compound’s spectrum have a difference in mass that meets any of the delta
masses in the delta mass filter, the compound will be displayed.
For both types of mass filters, a calculator is provided for calculating the exact mass of
the ion or delta mass. Example inputs for decane and its 13C isotope are shown below
to demonstrate the format required for formula input:
– For decane (C10H12), the input into the calculator should be as follows:
C10H22
– For the 13C isotope of decane (C913CH22), the input into the calculator
should be as follows (note the space required before and after the 13C
isotope): C9 C(13) H22.

Workflow
Figure 25 demonstrates how the mass delta calculator calculates the mass difference
of the two major chlorine isotopes that were used in the filter shown in Figure 23.
Figure 25. Using the Mass Delta Calculator
• Only Show Peaks with Library Hits — Select to filter the compound list only on
those compounds which have identifications associated with them. Additional
filtering based on total score, (R)HRF score, and R(SI) thresholds can be set.
Note If a sample has been processed using Reverse Search, then the threshold
values entered for the (R)HRF and (R)SI apply to the reverse search score for these
parameters. Otherwise these threshold values apply to the forward search values.
Further filtering based on the identified compound’s formula, CAS number, matched
compound name, library hit name, or synonym list names can be applied.
Library hit filters apply to the peak list only and depend on whether any of the peak's
associated library hits meet the specified criteria. For example, if no library hits for a
compound meet the search index threshold, then the compound does not appear in
the peak list. If one or more library hits for a compound meet the threshold, the
compound appears in the peak list, and all of its library hits appear regardless of
whether they meet the threshold.
Removing Unwanted Peaks
 To remove unwanted peaks from processed data
1. Click the Remove Peaks icon from the Peak List. See Figure 26.

Figure 26. Locating the Remove Peaks Icon
Remove Peaks
Icon
2. To remove the selected compound, select Remove Current Peak.

3. If a filter is applied, select Remove All Peaks Not Matching Current Filter. After selecting this,
compounds that have been removed by the applied filter do not reappear when the filter is
removed. This action can be performed on the selected sample, or on all samples in the batch.
4. To reverse actions to remove peaks, select Restore All Peaks. This action can be performed on the
selected sample, or on all samples in the batch.
Cross-Sample Compound Alignment (Optional)

After your data are processed, you can group compounds across the batch with similar spectra and
retention times if required for your analysis. This is useful if you want to compare the response of
compounds across samples in the batch. Once they are aligned, all the samples have the same list of
compounds, which makes it easy to compare them. See Figure 42.
 To align the compounds by retention times
1. In the Sample List in Sample View, check samples as Inclusion or Exclusion samples as shown in
Figure 27. Inclusion and exclusion samples will be explained in the following sections.
Workflow
Figure 27. Setting Samples to Inclusion or Exclusion
2. Select View > RT Alignment View. See Figure 28.

Figure 28. Opening the RT Alignment View
3. As shown in Figure 29, click the processing icon. Under RT Align Peaks select either All,
Inclusion, Exclusion or Modified Inclusion. These selections are explained below:
• All — Selecting All will look for all peaks detected in each processed sample in all other
processed samples.
• Inclusion — Selecting Inclusion will look for only those compounds detected in samples
marked inclusion samples in all other processed samples.
• Exclusion — Selecting Exclusion will look for all peaks detected in each processed sample
except for those peaks also found in samples that are marked exclusion samples. An example of
a sample type you might want to mark as an exclusion sample is a solvent blank, so that all
peaks found in the solvent blank are removed from the batch’s cross sample peak table.
• Modified Inclusion — Selecting Modified Inclusion will RT align those compounds
detected in samples marked inclusion samples, except for those compounds also detected in
exclusion samples.

Workflow
Figure 29. Aligning Compounds by Retention Times
Note This step might take a significant amount of time depending on the batch size.
Large numbers of files with many compounds also consume a lot of memory. You might
need to break a large batch into several smaller batches.
4. The Deconvolution Plugin Processing window opens. The green bars show the status of the RT
alignment process. See Figure 30.
Figure 30. Aligning Compounds in Batch by Retention Times
5. The compounds are now aligned by retention times. See Figure 31.

Workflow
Note RT Alignment View contains similar information to Sample View, with the addition of
displaying Peak Areas of compounds across samples in the batch. It also contains the Average Score
of a compound, which is the weighted average by intensity of a compound’s match score across the
batch. See Figure 31.
Figure 31. Compounds Aligned by Retention Times

Clear RT Average Score
Alignment Icon
Note Once compounds are aligned by retention time, certain features of the plugin are no
longer available. This is because each sample is no longer independent, and changing one
affects the entire batch.To use the disabled features, you must first clear RT alignment. See
Figure 31. Clearing RT alignment returns the batch to its state prior to RT alignment
processing.
Exporting Library Hits to the NIST Library

This section contains instructions for exporting the accurate-mass deconvoluted spectra from an
acquired compound to a .msp file for import into the NIST MS Search program.
 To export library hits to the NIST Library
1. From the Peak Identification window in Sample View, click the Export to NIST Library icon.
See Figure 32.

Workflow
Figure 32. Locating the Export to NIST Library Icon

Export to NIST
Icon
2. Select either Without Annotations to export spectra without additional comments, or With
Annotations to add additional information to the spectrum export.
3. If With Annotations is selected, the Annotations Editor window appears. See Figure 33
Figure 33. Viewing the Annotations Editor
4. You can enter text annotations under General, Sample, and Library Comments. The plugin
remembers annotations entered under General for all future spectrum exporting and those entered
under Sample for future spectrum exporting for the selected sample. The plugin remembers

Workflow
annotations entered under Library Comments only when exporting the spectrum from the
selected compound.
5. You can annotate spectra in custom fields or the general text field available in NIST formatted
spectra. In order to correctly visualize custom fields imported into the NIST MS Search program,
perform the following procedure.
6. After entering the Custom Field Names, click the Copy Custom Fields to Clipboard button in the
top left corner. See Figure 34.
Figure 34. Copying Custom Fields to the Annotations Clipboard
Copy Custom
Fields to
Clipboard Icon
7. In the NIST MS Search program, open the Comment Field Options. See Figure 35.
Figure 35. Opening the Comment Field Options
8. Overwrite the existing text with the comment fields copied to the clipboard. See Figure 36. Now
the NIST MS Search program recognizes these fields as custom fields when importing spectra.

Workflow
Figure 36. Overwriting Existing Text with Comments
9. Follow the procedure for importing the .msp file that was exported from the deconvolution
plugin, as described in the NIST MS Search help.
10. Your custom fields should now appear in the imported spectra. See Figure 37.
Figure 37. Viewing Custom Fields in Imported Spectra
Importing Results into TraceFinder Software

 To import results into the TraceFinder software application
1. First, process all the samples in the Deconvolution Plugin application. See Processing the Data.
2. The Deconvolution Plugin application allows users to set the mass tolerance for high-resolution
Orbitrap data and uses a mass tolerance of 500 mmu for unit resolution nominal mass data. You
must change the mass tolerance in the Master Method in TraceFinder software. Go to Analysis >
Local Method > Processing to change the mass tolerance. See Figure 38.

Workflow
Figure 38. Setting the Mass Tolerance

Mass Tolerance
Note The default number of library matches in the TraceFinder application is three,
which might not be enough for your needs. To increase the number of library matches,
before running samples, open the Analysis panel in the TraceFinder application, and go
to Processing > Peak Settings. Change the Number of Top Library Matches to the
number you would like TraceFinder to keep.
3. Ensure that under Search Options, only Library Search is selected. Other search options aren’t
supported in this version of the plugin.

Workflow
Figure 39. Setting the Search Options

Library Search Only Checked
4. Next, under Local Method in the side menu of TraceFinder software, click Peak Detection
Settings and change the Detection Algorithm to Avalon. Next, change the Detection Method to
Nearest RT. TraceFinder software peak detects based on the RT and m/z reported by the plugin.
To accurately pick the correct peak in a busy chromatogram, TraceFinder software must be set for
Nearest RT rather than Highest Peak. Otherwise, TraceFinder software may find a neighboring
isomer or other similar peak if it is bigger than the real peak. Also, it’s recommended to set Avalon
parameters as seen in Figure 40 so they closely match the peak detection settings used in the
plugin.
Note Setting the peak detection settings to Avalon, Nearest RT, smoothing to 9, and mass
tolerance to the same value set in the Deconvolution Plugin application processing
settings makes the area and height results in TraceFinder software and the plugin more
consistent. There still might be differences in the results.

Workflow
Figure 40. Changing the Detection Method in TraceFinder
5. Go to Batch View on the left side menu in TraceFinder software.

6. Click the Submit Batch icon to import the samples into TraceFinder. See Figure 41.
7. The Submit Options dialog box opens. See Figure 41. Submit the batch for processing with
Unknown Screening settings selected within TraceFinder software so that they match the
processing in the Deconvolution Plugin application. The allowed setting combinations are:
• If only peak detection was applied to samples in the plugin, select only Peak Detect in
TraceFinder software.
• If peak detection and library search were applied to samples in the plugin, select Peak Detect
and Identify in TraceFinder software.
• If peak detection, library search, and RT alignment were applied to samples in the plugin,
select Peak Detect, Identify, and With RT Alignment in TraceFinder software.
• If peak detection and RT alignment were applied to samples in the plugin, select Peak Detect
and With RT Alignment in TraceFinder software.
No other combinations are allowed.
Note TraceFinder software only allows one mass tolerance to be applied to a batch.

Workflow
Figure 41. Setting Submit Options

Submit Batch
Icon
Note TraceFinder software uses retention time and base peak mass provided by the plugin
application to peak detect compounds displayed in Unknown Screening View. For this
reason, it might take a significant amount of time to import compounds into the
TraceFinder Unknown Screening View.
8. The results appear in the TraceFinder unknowns view. See Figure 42.
Figure 42. Viewing the Results in the TraceFinder Unknowns View

Workflow
9. Use the View menu in TraceFinder data review to choose what parameters you want to see on
screen for your data review.
Figure 43. Selecting Data Review Parameters in TraceFinder Software
Note For detailed information on using TraceFinder Unknowns View, see the TraceFinder user
documentation or Help.
Creating a Report
After you have viewed the results of your analysis in TraceFinder software, you can export the data for
further analysis; for example, into a statistical software package. The Deconvolution Plugin application
supplies a sample report template for creating cross sample peak tables in a format that you can import
into these applications. These templates are installed automatically with the plugin.
 To create a report using an installed template
1. Check the compounds you want to export in the Selected column in the Cross Sample Peak List
tab in the TraceFinder Unknown View. No compounds are checked by default. See Figure 44.
Note This step only applies if you are using one of the Cross Sample Peak Table Report templates.

Workflow
Figure 44. Checking Compounds to Export in TraceFinder Unknowns View

Selected
Column
2. In TraceFinder software, go to Analysis > Report View > Local Method > Reports.
3. The Report View page opens. See Figure 45.
Figure 45. Report View Page
4. Choose the report template you want (Cross Sample Peak Table 1 or Cross Sample Peak Table 2)
from the Template list.

Workflow
5. Check the correct box for the format you want for your report. The choices are PDF, CSV, Excel,
or Print. Export as CSV or Excel for import into third-party software packages.
6. Click Generate to generate your report.

Workflow

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Deconvolution Plugin for

ISQ LT, TSQ 8000 Evo, and

1R120706-0006 Revision B February 2016

Thermo Fisher Scientific Inc. All rights reserved.

Software version: Exactive Series 2.7 or later; TraceFinder 4.0 or later

For Research Use Only. Not for use in diagnostic procedures.

Deconvolution Plugin Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

 To view the product manuals

Go to Start > Programs > Thermo Exactive Series > Manuals.

Thermo Scientific Deconvolution Plug In Software Manual (P/N 1R120706-0006, Revsion B) v

 To view the product manuals

Open the Manuals folder on your desktop.

 To view the product manuals

Open the Manuals folder on your desktop.

For access to the application Help, follow this procedure.

 To view application-specific Help

For more information, visit www.thermoscientific.com.

vi Deconvolution Plug In Software Manual (P/N 1R120706-0006, Revsion B) Thermo Scientific

System Minimum requirements

Cautions and Special Notices

CAUTION Highlights hazards to humans, property, or the environment. Each CAUTION

IMPORTANT Highlights information necessary to prevent damage to software, loss of

Note Highlights information of general interest.

Tip Highlights helpful information that can make a task easier.

 To find out more about our products

Go to www.thermoscientific.com/en/products/mass-spectrometry.html for information

 To get local contact information for sales or service

 To suggest changes to documentation or to Help

• Fill out a reader survey online at www.surveymonkey.com/s/PQM6P62 or send an

Setting Up the Deconvolution Plugin

Thermo Scientific Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) 1

5. Restart the TraceFinder application to view the plugin.

2 Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) Thermo Scientific

Figure 3 shows the Sample View of the Deconvolution Plugin application.

Thermo Scientific Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) 3

4 Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) Thermo Scientific

• Matched Compound — The primary name of a compound in the selected library.

Thermo Scientific Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) 5

Total Ion Chromatogram

Active Ion Trace

Active Ion Trace

6 Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) Thermo Scientific

• Measured m/z — The measured mass-to-charge ratio of the active mass.

Thermo Scientific Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) 7

Figure 9. Spectra Window

 To acquire data in TraceFinder software

1. First, go to Method Development in TraceFinder and create an unknown screening

8 Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) Thermo Scientific

Figure 10. Creating a Batch

Thermo Scientific Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) 9

Figure 11. Naming a Batch

10 Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) Thermo Scientific

Processing the Data

Functional Description of Processing Parameters

 To access the processing parameters in the Deconvolution Plugin application

2. The Processing Parameters window opens. See Figure 14.

Thermo Scientific Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) 11

Figure 14. Viewing the Processing Parameters

12 Deconvolution Plugin Software Manual (P/N 1R120706-0006, Revision B) Thermo Scientific