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SimLipid

3.5

Manual

PREMIER

Biosoft International

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Palo Alto, CA 94303-4504

Tel: (650) 856-2703

FAX: (650) 843-1250

E-mail: [email protected]

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Copyright © 2003 by PREMIER Biosoft International. All Rights Reserved.

Information in this manual may change without notice and does not represent a commitment on the part of PREMIER Biosoft International.

The software described in this manual is provided by PREMIER Biosoft International under a License Agreement. The software may be used only in accordance with the terms of the agreement.

PREMIER Biosoft International ("Premier") claims copyright to this program and documentation as an unpublished work. Claim of copyright does not imply waiver of Premier's other rights.

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For more information on SimLipid and product updates, visit the PREMIER Biosoft web site at http://www.PremierBiosoft.com

SimLipid 3.5 i

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INTRODUCTION .................................................................................................. 1

CREATE AND MANAGE PROJECT ................................................................... 2

CREATE NEW PEAK LIST.................................................................................. 3

OPEN PEAK LIST DATA FILES.......................................................................... 3

AVERAGE SCANS ............................................................................................ 12

SORT PEAK LIST DATA................................................................................... 13

MS SEARCH...................................................................................................... 16

MASS SPECTRUM ANNOTATION ................................................................... 20

PLOT MASS SPECTRUM ................................................................................. 21

ADD TEMPLATE FOR SEARCH PARAMETERS............................................. 21

MS/ MS SEARCH............................................................................................... 23

MS/MS ANNOTATION....................................................................................... 27

EXPORT ANNOTATION.................................................................................... 28

PLOT MASS SPECTRUM ................................................................................. 28

COMPREHENSIVE REPORT OF LIPIDS IDENTIFIED USING MS AND COMPLEMENTARY MS/MS DATA................................................................... 28

COMPARATIVE REPORT TO VALIDATE LIPIDS IDENTIFIED IN MS OR MS/MS DATA FROM VARIABLE ION MODES................................................. 31

COMPARE PROFILE MS SPECTRA ................................................................ 33

COMPARE MS/MS RESULTS BY ALIGNING RETENTION TIME ................... 34

HIGH THROUGHPUT LIPID SEARCH .............................................................. 36

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GENERATE REPORT........................................................................................ 43

EXPORT RESULTS........................................................................................... 44

EXPORT RESULTS TO AGILENT'S CEF FILE ................................................ 45

FIND LIPIDS....................................................................................................... 46

LIPID QUANTITATION ...................................................................................... 48

SYSTEM SPECIFICATIONS.............................................................................. 53

COPYRIGHT ...................................................................................................... 54

REFERENCES................................................................................................... 54

SimLipid 3.5 1

Introduction

Mass Spectrometry with its unique sensitivity, high resolution, and versatility has gained a

growing role in lipidome analysis. SimLipid is a comprehensive lipidome MS, MS/MS and MSE

data analysis software, enables you to identify lipids using mass spectrometric data, calculates

isotope percentages of identified lipids, corrects the experimental profile and identifies unknown

lipid structures using precursor and product ion data.

The SimLipid structure database includes 8 lipid categories and 36224 lipid species along with

their additional biological information such as lipid abbreviation, systematic name, mass,

composition and links to the other databases. The program accepts experimental MS data and

MS/ MS data (m/z and intensity values) in text, MS Excel, mzData, mzXML, Waters MSE, AB

SCIEX binary file formats(*.t2d and *.Wiff files), Bruker Daltonics (baf, yep and fid), Thermo

Scientific (.RAW) and Agilent (.cef) data files. SimLipid annotates mass spectra with the lipids

identified for MS as well as MS/MS data.

High Throughput MS Lipid Search

SimLipid enables high throughput lipid profiling from multiple MS peak list data by searching lipid

precursor ions against the known lipid structures available in the SimLipid database for Fatty

acyls, Glycerophospholipids, Glycerolipids, Polyketides, Prenols, Saccharolipids, Sphingolipids

and Sterols. Users can also trigger product ion data analysis from an MS lipid profile. SimLipid

offers an intuitive user interface to perform such analysis with ease.

Lipid Structural Elucidation using MS/MS Data

SimLipid enables you to analyze MS/MS and MSE data for Glycerophospholipids, Glycerolipids,

Sphingolipids, Prenol Lipids and Saccharolipids for 300 scans in batch mode. High resolution

accurate mass data with error tolerance between 1-20 parts per million (ppm) and 0.1 to 2000

milliDaltons (mDa) can also be analyzed. The results are sorted using an innovating ranking

algorithm that maps the degree of proximity of theoretical lipids with experimental data and the

two dimensional structure of the lipids identified is displayed.

High Throughput Lipid Profiling and Quantitation

SimLipid enables relative quantitation of lipids corresponding to the observed peaks on MS

spectra. Quantification is done by normalizing the intensity values based on the observed

intensity of an internal standard. To enable quantification of lipids from low resolution spectra, the

program corrects the intensities for isotopic overlapping of peaks. This facilitates accurate

quantification of lipids from biological mixtures. The quantified data along with other information

such as peak m/z, molecular formula of lipids, corrected, uncorrected and normalized intensities

in different samples can be exported to a spreadsheet compatible .csv and .xls formats.

SimLipid 3.5 2

Mass Spectra Annotation with Identified Lipids

SimLipid can annotate mass spectra with the lipids identified for MS, MS/MS as well as MSE

data.

This helps in interpreting mass spectra by highlighting the experimental m/z values that match

those of theoretical lipid structures from the product database. Each matched peak can be

annotated with either the lipid fragment name or its corresponding lipid chemical formula. You can

export the annotated spectra as image files to MS PowerPoint, PNG and JPEG files to facilitate

information sharing amongst research groups.

The annotated mass spectra can be adjudged/re-sized either to fit on a page or based on the

area of interest. A user can zoom in/out into a specific plot location using the mouse cursor or by

specifying the m/z range. Further, users can export the annotated spectra as images (in PNG and

JPEG formats) that can be included in MS PowerPoint, facilitating information sharing amongst

research groups.

Create and Manage Project

SimLipid enables organizing your work by creating separate projects. You can create any number

of projects or edit an already existing project. You can start working with SimLipid by creating a

new project. One project can be opened at a time.

Create a New Project

1. Choose File > New > Project or click from the tool bar. Enter a project name

2. By default, a project is created in the SL Projects folder in the product installation folder

(C:\Program Files\SimLipid\SLProjects). You can browse the New Project dialog and create a

project at any other location on a local drive.

Open Project

1. Choose File> Open Project or click from the tool bar. Browse and select the ..... file and

Click Open.

2. You can also open a project by selecting the project displayed in the history list displayed at

the bottom of the file menu. SimLipid displays the most recent project in the history list.

Close Project

1. Choose File > Close Project. This will close the project.

2. Choose File > Exit. This will close the project and exit the program.

3. An already open project is closed automatically when you open another project.

SimLipid 3.5 3

Create New Peak List

SimLipid enables entering of m/z values and intensity manually.

1. Choose File>New> New Peak List or click from the tool bar.

2. A Create New Peak List dialog launches with two columns for m/z and intensity where the

values can be entered manually or copy-pasted from another source.

3. Click OK . A new MS Profile is created in the project.

Open Peak List Data Files

SimLipid accepts MS Profiles in text, MS Excel, mzXML, mzData formats. Support for AB SCIEX

binary file formats, *.t2d and *.Wiff files, Bruker Daltonics baf, yep and fid file format, Thermo

Scientific (.raw) , Agilent(.cef) data files and Waters MSE data files is also available. The program

is capable of importing data from complete chromatogram runs i.e., up to 20,000 scans in a single

project.

1. Go to File> Open Peak List Data has the following options:

a) mzData/ mzXML

b) Text/ Excel

c) Waters MSE Peak List Data

d) Bruker Daltonics Peak List Data

e) Thermo Scientific Data File

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f) AB SCIEX Data File

g) Agilent CEF Data Files

1. To open any of the supported MS data files, go to File>Open Peak List Data> ...or click

from the toolbar:

2. Load Relative Intensity = ...: This enables uploading peaks above a threshold relative

intensity ( % of maximum intensity) value.

Import mzData/ mzXML Files

1. Choose File>Open Peak List Data> mzXML/mzData

2. Browse to the mzData/mzXML Data File and click Open.

3 The Load Scan dialog launches. You can specify the Precursor ion m/z and charge state for

the scans. Enter the m/z and charge state and click Apply. Select specific scan numbers for

which you would like to search Lipid structures.

4. Click Load Scan. Peak list data for the selected scan numbers is loaded.

Import Text/ Excel Files

1. Choose File>Open Peak List Data>Text/Excel

2. In the Open File dialog, browse and select the files. An Open Peak List Data dialog is

launched. Specify the Precursor ion m/z and charge state values and click Apply. The profiles

with missing values is updated.

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3. Also specify whether the data belongs to MS/ MS by selecting the check box in MS/ MS Data

column. SimLipid can also import Waters' direct infusion data in .xls format along with Precursor

Ion Scan (PIS) and Neutral Loss Scan (NLS) information

4. To edit the values for a particular scan, double click the cell and enter the appropriate value.

Import Waters Peak List Data Files

1. To open Waters MSE and DDA data files, go to File>Open Peak List Data> Waters MSE ...or

click from the toolbar.

2. An Open Waters Peak List Data dialog is launched.

3. Load Relative Intensity =...: This enables uploading peaks above a threshold relative intensity

( % of maximum intensity) value.

4. Browse to the data file and click Open. Peak list data for the selected data file is loaded.

5. In order to facilitate MS Search using precursor m/z values from the MSE data file, SimLipid

creates a peak list profile with the name @Precursors.

For example, from the above example, MSe DV_Txt-OP_Pos_all_120910 where MSe DV_Txt-

OP_Pos_all_120910 is the file name. The peak list profile contains only the Precursor m/z values,

corresponding intensity values and charge states.

6. Peak list profile corresponding to product ions for each reported precursor m/z is created with

the name <Precursor m/z><Retention Time>. For example, the profile 496.309_1.9578 is the

SimLipid 3.5 6

peak list data containing the product ions of precursor m/z 496.309 and retention time 1.9578.

7. MS Search is enabled for <Precursor m/z><Retention Time> and MS/ MS Search for product

ions.

Import Bruker Daltonics Native Data Files

SimLipid can import native data files of Bruker Daltonics mass spectrometers in FID, BAF and

YEP file formats. CompassXtract is required to be installed on the same computer as SimLipid for

the import. CompassXtract can be downloaded from Bruker's website (http://bruker-daltonics-

compassxtract.software.informer.com/download/). After the installation is complete, please follow

the steps below:

Steps to import BAF/YEP files:

1. Choose File>Open Peak List Data> Bruker Daltonics or click from the tool bar for

BAF,/YEP.

2. Browse to the Bruker Data File (BAF, YEP) and click Open. Load Scan dialog launches.

3. If BAF/ YEP file is selected, specify Data Type and Peak Shoulder Intensity. SimLipid peak

picking algorithm enables you to detect/ignore peak shoulders if the slope of such peak is

greater/less than a specified threshold value as the percentage of the maximum slope on the

peak. For BAF/ YEP, default threshold value is 1.

4 Now specify the Precursor m/z and Charge State for the profiles.

5. Specify Peak Shoulder Intensity. The minimum value for Peak Shoulder Intensity is 0 and

maximum is 100. Select the scans to be loaded and click Load Scan. Selected scans are loaded

in MS Profile/ MS/ MS Profile tab.

Steps to import FID files:

SimLipid 3.5 7

1. Choose File>Open Peak List Data> Bruker Daltonics or click from the tool bar for FID

file.

2. Browse to the Bruker Data File (FID) and click Open. Load Scan dialog launches.

3. If FID file is selected, specify File Type, Data Type and Peak Shoulder Intensity.

4. If FID file is selected, specify Data Type and Peak Shoulder Intensity. For FID, default

threshold value is

5. .Now specify the Precursor m/z and Charge State for the profiles.

6. Specify Peak Shoulder Intensity. The minimum value for Peak Shoulder Intensity is 0 and

maximum is 100.

7. Select the scans to be loaded and click Load Scan. Selected scans are loaded in MS Profile/

MS/ MS Profile tab.

Import Thermo Scientific Native Data Files

SimLipid requires Thermo's MSFileReader to load Thermo Scientific native data files. It can be

installed from a self-extracting archive, available for download from Thermo Scientific website

(http://sjsupport.thermofinnigan.com/public/detail.asp?id=703)

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The setup starts automatically and leads the user through the installation process. Once

MSFileReader is successfully installed, follow the steps below to import RAW files:

1. Choose File>Open Peak List Data>Thermo Scientific Data File or click from the

toolbar.

2. Browse to the data file(s) (.RAW) and click Open.

3. Browse to the Bruker Data File (BAF, YEP) and click Open. Load Scan dialog launches.

4. Data can be extracted either in profile or centroid mode. SimLipid automatically reads the data

mode.

5. The Profile mode enables loading either peak tops or whole data.

6. The Centroid mode enables loading the data after centroiding the peaks within a specific peak

width. The peak width can be specified between 0 and 1.

7. Now select the specific scan numbers for which you would like to perform a search.

8. Click Load Scan to load the selected scans in the program for further analysis.

Import AB SCIEX Native Data Files

One of the following tools should be installed on the same computer as SimLipid to load *.Wiff

files.

a) Analyst® 1.5.1 Software

b) Analyst® TF

c) Analyst® QS 2.0

The tools are available for download from AB SCIEX website

(http://www.absciex.com/downloads/software-downloads)

Import *.Wiff files in SimLipid

1. Go to File>Open Peak List Data> AB SCIEX Data File, browse and select the file. It opens in

the currently open project.

2. Select the cycles/experiments to be loaded from each sample using standard keystrokes

(Ctrl+Click).

Confirm the selection by using the >> button. The selected data files are displayed in the table to

the right.

3. Unchecking the Load Scans with precursor ion m/z>0 option enables you to load all the

scans containing data with or with out precursor ion m/z information.

4. Clicking OK launches the Select Scan dialog.

SimLipid 3.5 9

5. Enter the precursor ion m/z and charge state values of the corresponding cycles and click

Apply. The scans with missing values are updated.

6. To edit the values for a particular scan, double click the cell and enter the correct value.

7. Select the scans to be loaded by checking the boxes under the MS/MS data column and

clicking Load Scan. The selected data is loaded in the program.

Import*.t2d files in SimLipid:

1.Go to File>Open Peak List Data> AB SCIEX Data File or click from the tool bar. Browse

and select the file. It opens in the currently open project.

2. The Load Profiles dialog launches. Enter the precursor ion m/z and charge state values of the

corresponding cycles and click Apply. The scans with missing values are updated. To edit the

values for a particular scan, double click the cell and enter the correct value.

3. Select the scans to be loaded by checking the boxes under the MS/MS data column and

clicking Load.

SimLipid 3.5 10

Import Agilent CEF file support

A) Import up to 3 CEF files:

1. Choose File> Open Peak List Data> Agilent CEF Data File> Import up to 3 files or click

from the toolbar.

2. Browse the file location, select the CEF Data Files and click Open. Load Compounds dialog

launches.

3. The data from the files selected is shown under different tabs. You have the option to see all

compounds of a specific MS level and specify the Precursor Ion m/z and Charge State if

missing.

4. Specify Range dialog provides an option to filter compounds based on Retention Time,

Compound IDs or Precursor m/z values. Further, MS and MS/MS data can be filtered based on

m/z range and threshold Intensity values. SimLipid assigns the ion species of the MS/MS

precursor m/z by probing its parent MS data. In order to match the MS/MS precursor m/z value

against MS data, specify the m/z Precision (decimal points) and Error Tolerance parameters.

SimLipid 3.5 11

5. Click Load Selection. Peak list data for the selected compound numbers is loaded.

B) Import up to 100 CEF files:

1. For loading up to 100 CEF files, go to File> Open Peak List Data> Agilent CEF Data File>

Import files in batch mode.

2. File chooser dialog launches. Browse to the Agilent CEF Data File location, select up to 100

CEF files and click Open.

3. Specify Range dialog appears. Specify Range dialog provides an option to filter compounds

based on Retention Time, compound IDs or Precursor m/z values. Further, MS and MS/MS

data can be filtered based on m/z range and threshold Intensity values. SimLipid assigns the ion

species of the MS/MS precursor m/z by probing its parent MS data. In order to match the MS/MS

precursor m/z value against the MS data, specify the m/z Precision (decimal points) and Error

Tolerance parameters.

4) Click OK. Peak list data for the selected compounds are loaded.

SimLipid 3.5 12

Average Scans

This feature enables users to create a peak list by averaging data from multiple scans.

To average the scans:

1. Go to Tools> Average Scans or click from the tool bar.

2 Average Scans dialog is launched.

.

.

3. Select the data type as either MS Data or MS/ MS Data.

4. All the scans are displayed in the table below

SimLipid 3.5 13

5. Select the scans to be averaged using the following options:

a) Scan No.: Specify the range of the scan numbers to be averaged.

b) Retention Time: Specify the range of retention time to be averaged.

c) Precursor m/z: Specify the range of precursor m/z to be averaged.

6. To ignore scans without retention time information, check option Ignore the row for which

Retention Time is not available.

7. Select Data Pre-processing options

a) m/z Options:

i) Consider m/z peaks observed in all the samples: Only those m/z values which are observed in

every MS data file/scan are considered. If left unchecked, then all the m/z values observed in all

the selected MS data files are considered.

Note: If an m/z is not observed in the sample, its intensity is considered as zero.

ii) m/z precision (decimal points): You can specify the data precision. By default, it is set as 4

which mean that the data is rounded off to its first decimal place. Users can set any value

between 0 and 4.

Example: m/z precision (decimal points): 0. If 701.5 is the observed m/z value, then SimLipid

rounds it up to 702.

b) Intensity Options:

i) Multiple peaks with same m/z within round off limits: You can either select the Average

Intensity or Highest Intensity option. If Average Intensity is selected, SimLipid calculates the

average intensity from multiple peaks which have the same m/z value (after rounding them off). If

Highest Intensity is selected, SimLipid selects the highest intensity value for any given m/z.

ii) Threshold Intensity: For Threshold Intensity, you can select either Relative or Value. SimLipid

ignores all the peaks below the specified threshold.

8. Click Average Scans, averaged data is displayed in the project management panel as a

separate node.

Sort Peak List Data

SimLipid enables sorting of the peak list data on the basis of Precursor Ion m/z, Charge State,

Retention Time, Intensity and Drift Time for the file formats supported. For mzXML and mzData

files, sorting can, in addition, be done on Scan No, Parent Scan No and MS Level. This facilitates

convenient investigation of data scattered across different scans or profiles by allowing triggering

of MS/MS analysis from a peak of interest.

Steps to perform sorting:

SimLipid 3.5 14

1. Go to Edit> Sort Peak Lists or click on the Sort Peak List icon from the toolbar.

2. Depending on the type of file selected (mzXML/ mzData, Waters MSE, text/ Excel. manual

entry), the loaded files are displayed in the drop down for file selection. For mzXML, mzData and

Waters MSE files, one file can be selected for sorting.

3. For text, Excel and manual entry, multiple files can be selected.

4. Sort Peak List dialog for mzXML and mzData files:

5. Sort Peak List dialog for Text, Excel and Waters MSE files:

SimLipid 3.5 15

6. File(s) are selected from the Select Profile drop down. After clicking OK, the selected files are

displayed in the table below.

7. On clicking Edit, the Edit dialog launches. Enter the precursor ion m/z, charge state, retention

time and drift time. On clicking

Default, the default parameters are displayed Click OK.

8. Enter the criteria under Sort by, Then by and Order options in the drop down and click Sort.

SimLipid 3.5 16

On clicking Rearrange Profiles, sorted profiles are loaded in the Project Management panel.

Sort by refers to the primary sorting information on which the peak lists are to be sorted. Then by

refers the secondary information using which sorting is to be performed. You can also select

None in this drop down. The Order option enables displaying data in Ascending or Descending

order.

MS Search

SimLipid accepts experimental m/z and intensity values for profiling lipids by searching precursor

ions against the known lipid structures available in SimLipid database.

1. MS data selected in the project management panel is displayed in the MS profile tab.

2. Before performing an MS Search, No. of Hits in the MS Profile tab is reported as 0.

3. Click from the tool bar or go to Analyze> MS Search.

4. MS Search dialog launches. You can specify the Template Name, Error Tolerance, Threshold

Relative Intensity (%), Polarity and Ion Mode. SimLipid supports [M+H], [M+NH4], [M+Na],

[M+C5H12N], [M+Li], [M+K] ions in positive ion mode and [M-H], [M+AcO], [M+Cl], [M-CH3],

[M+OAcO]and [M+HCOO] in negative ion mode.

5. Enter the required parameters and click Search.

6. After performing a search, the number of lipids obtained corresponding to a particular peak is

SimLipid 3.5 17

displayed in the column No. of Hits. If no match is found, it is displayed as 0.

7. Select a row in MS profile tab as per the peak of interest and switch to the Search Results tab.

8. Resultant lipids for the selected m/z are highlighted in the Search Results tab. You can also

select to view the distinct chemical formula or m/z value for a reported peak.

SimLipid 3.5 18

Trigger MS/ MS Search for a Single Precursor Ion

1. To trigger an MS/MS Search, go to Analyze> click MS/ MS Search.

2. For MSE

data files, the corresponding MS/ MS profiles are detected by the program and MS/

MS Search parameter dialog is launched. For other file formats, Select MS/ MS Profile dialog is

launched. All the MS/ MS profiles are displayed here. Select a profile and click OK.

SimLipid 3.5 19

3. MS/MS Search dialog launches.

4. Enter MS/ MS Search parameters:

a) Template Name: You can create templates of search parameters which are to used

repeatedly for analysis. The templates so created can be retrieved by choosing the Template

Name in the MS or MS/MS dialog.

b) Precursor Ion m/z: This is the value of the m/z observed on MS spectrum to be subjected to

MS/MS fragmentation. Precursor Ion m/z information is automatically read from mzXML, mzData

and Waters MSE

files. For .txt, .xls files and manually entered peak list data, it should be specified

while importing the data.

c) Precursor Ion Error Tolerance: This is the allowable range of deviation of theoretical m/z

values of candidate lipids from observed precursor m/z value. You can specify the Precursor Ion

Error Tolerance in Dalton (Da), milli Dalton(mDa) and in parts per million (ppm). The range of

Dalton is 0.0001 to 2.0, ppm is 1 to 20 and mDa is 0.1 to 2000.

d) Charge State: This is the charge state of the observed precursor m/z value. It is automatically

read from mzXML, mzData and Waters MSE

files. For .txt, .xls files and manually entered peak list

data, it should be specified while importing the data, between 1-9.

SimLipid 3.5 20

e) MS/ MS Error Tolerance: This is the allowable range of deviation of theoretical m/z values of

lipid fragments from observed product ion m/z values. You can specify MS/ MS Error Tolerance in

Dalton, mDa or in ppm. The range of Dalton is 0.0001 to 2.0, ppm is 1 to 20 and mDa is 0.1 to

2000.

f) Polarity: If the observed precursor ion is positively charged, choose positive and if it is

negatively charged, choose negative.

g) Ion Mode: Select an ion mode from the list. SimLipid supports [M+H], [M+NH4], [M+Na],

[M+C5H12N], [M+Li], [M+K] ions in positive ion mode and [M-H], [M+AcO], [M+Cl], [M-CH3] and

[M+OAcO] in negative ion mode.

h) Lipid Category: You can specify Lipid Categories, Lipid Class and Sub Class to filter the

search results. SimLipid will restrict the search of candidate lipids to the specified sub classes.

5. After entering all the parameters, click Search. A ranked list of resultant lipids is displayed in

the Search Results tab.

Export Selected Results

1. On clicking Export Selected Lipids, Export Selected Results dialog is launched.

2. You can export the results in Excel, CSV or HTML file formats.

a) Lipid Selection:

Lipids for selected m/z are displayed here using the check boxes on the left of each.

b) Lipid Options:

You can select the information to be exported. For example m/z, Lipid Abbreviation/Common

Name, Intensity, Delta Mass, Ion Type, Category, Lipid Structure, Retention Time and Drift Time.

c) Output Options:

File Type: The results can be exported in Excel, CSV or HTML file formats for sharing information

with colleagues or publishing results. .

Mass Spectrum Annotation

SimLipid can annotate mass spectra using abbreviations of the lipid structures identified. It helps

in interpreting a mass spectra by highlighting the experimental m/z values that match those of

theoretical lipid structures from SimLipid database and generates an annotated spectrum.

Mass Annotation

1. Go to View>Mass Annotation. This option is enabled only when MS Lipid Search has been

performed.

2. In the Mass Annotation tab, m/z, intensity, lipid abbreviations and ion type are displayed.

SimLipid 3.5 21

Matched m/z values are highlighted in blue and are annotated with the lipids structure that

matches. On selecting a particular lipid and clicking View Lipid Structure, the structure is

displayed. Unmatched m/z values are displayed in yellow.

3. Export Annotation

Select lipids and click Export Annotation. Selected lipids are exported in HTML format

Plot Mass Spectrum

1. Select lipids to be plotted in the Mass Annotation tab and click Plot Spectrum.

2. A mass spectrum is displayed depicting lipid abbreviations on the selected peaks. The

annotated mass spectra can be re-sized either to fit a page or based on the area of interest, you

can zoom in/out into a specific plot locations or specify m/z range to display specific plot sections

for better analysis of the annotated spectra. . You can search a particular lipid by entering its m/z

or intensity value in the Lipid Information table and clicking the Search button The mass spectra

can be exported in PNG/JPEG file format.

Add Template for Search Parameters

SimLipid enables applying the same search parameters over different data set by the use of

templates. Users can Add/Remove/Edit a template of search parameters. The added template/s

can be retrieved by choosing the Template Name in the MS or MS/MS “Search” dialogs.

To add a template for MS Search

1) Go to Tools>Add Template> MS Search

2) Add MS Search Parameter Template dialog is opened. Specify the name and search

parameters. Click Add. The search template is added to the template list.

3) The added template/s can be retrieved by clicking on the Template Name in the MS Search

dialog.

To add a template for MS/MS Search

1) Go to Tools>Add Template> MS/MS Search

2) Add MS/MS Search Parameter Template dialog is opened. Specify the name and search


3) The added template/s can be retrieved by clicking on the Template Name in the MS> MS

Search dialog.

To add a template for Lipid Quantitation

1) Go to Tools>Add Template> Lipid Quantitation

SimLipid 3.5 22

2) Add Search Lipids Parameter Template dialog is opened. Specify the name and search


3) The added template/s can be retrieved by clicking on the Template Name in the Search Lipid

dialog.

Remove/Edit Template

Remove Template:

1) Go to Tools>Remove/Edit Template.

2) List of added templates are displayed. Select the template to be removed and press Remove.

The added template is removed.

Edit Template:

1) Go to Tools>Remove/Edit Template. Select the template to be edited and press Edit.

2) Change the necessary parameters and press Update.

Add Template for Search Parameters

SimLipid enables applying the same search parameters over different data set by the use of

templates. Users can Add/Remove/Edit a template of search parameters. The added template/s

can be retrieved by choosing the Template Name in the MS or MS/MS “Search” dialogs.

To add a template for MS Search

1) Go to Tools>Add Template> MS Search

2) Add MS Search Parameter Template dialog is opened. Specify the name and search


3) The added template/s can be retrieved by clicking on the Template Name in the MS Search

dialog.

To add a template for MS/MS Search

1) Go to Tools>Add Template> MS/MS Search

2) Add MS/MS Search Parameter Template dialog is opened. Specify the name and search


3) The added template/s can be retrieved by clicking on the Template Name in the MS> MS

Search dialog.

SimLipid 3.5 23

MS/ MS Search

SimLipid allows you to perform structural elucidation of lipids MS/MS data for

Glycerophospholipids [GP], Sphingolipids[SP], Glycerolipids [GL], Prenol Lipids [PR] and

Saccharolipids [SL].

SimLipid detects the MS level of peak list data files uploaded in mzXML, mzData and Waters MSe

file formats. The information is displayed as tool tip in the Project Management panel. For .txt, .xls

files and manually entered peak list data, MS/ MS Search module is activated only when the peak

list data is selected as MS/MS data while importing the file.

Steps to perform MS/ MS Search:

1. Go to Analyze>MS/MS search or click from the tool bar.

2. MS/MS Search dialog launches.

Precursor Ion m/z: This is the value of the m/z observed on MS spectrum which has been

subjected to MS/MS fragmentation. Precursor Ion m/z information is automatically read from

mzXML, mzData and Waters MSe files. For .txt, .xls files and manually entered peak list data, it

should be specified while importing the data.

Precursor Ion Error Tolerance: This is the allowable range of deviation of theoretical m/z values

SimLipid 3.5 24

of candidate lipids from observed precursor m/z value. You can specify the Precursor Ion Error

Tolerance in Dalton (Da), milli Dalton(mDa) and in parts per million (ppm). The range of Dalton is

0.0001 to 2.0, ppm is 1 to 20 and mDa is 0.1 to 2000.

Charge State: This is the charge state of the observed precursor m/z value. It is automatically

read from mzXML, mzData and Waters MSe files. For .txt, .xls files and manually entered peak list

data, it should be specified while importing the data, between 1-9.

MS/ MS Error Tolerance: This is the allowable range of deviation of theoretical m/z values of

lipid fragments from observed product ion m/z values. You can specify MS/ MS Error Tolerance in

Dalton, mDa or in ppm. The range of Dalton is 0.0001 to 2.0, ppm is 1 to 20 and mDa is 0.1 to

2000.

Polarity: If the observed precursor ion is positively charged, choose positive and if it is negatively

charged, choose negative.

Ion Mode: Select an ion mode from the list. SimLipid supports [M+H], [M+NH4], [M+Na],

[M+C5H12N], [M+Li], [M+K] ions in positive ion mode and [M-H], [M+AcO], [M+Cl], [M-CH3] and

[M+OAcO] in negative ion mode.

Lipid Category: You can specify Lipid Categories, Lipid Class and Sub Class to filter the search

results. SimLipid will restrict the search of candidate lipids to the specified sub classes.

3. After entering all the parameters, click Search. Resultants lipids are ranked on basis of

calculated score in the Search Results tab.

SimLipid 3.5 25

4. Search Results tab includes:

Lipid Rank: Rank of Lipids on basis of calculated relative scores using a proprietary ranking

algorithm.

Lipid ID: Identification number of Lipid in SimLipid database.

Chemical Composition: Molecular formula of lipid.

Experimental m/z: Experimental m/z value observed on the mass spectrum.

Theoretical m/z: Theoretical m/z calculated on basis of molecular formula of compounds. All the

values are based on monoisotopic mass of compounds.

SimLipid 3.5 26

Delta Mass: Delta Mass is the difference between theoretical m/z and m/z observed on

experimental spectra.

Score: Calculated relative scores based on proprietary lipid ranking algorithm. Lipid with highest

score is the most probable structure among the probable structures matching the precursor ion

m/z value.

5. On clicking Lipid Structure, lipid structure is displayed.

6. On clicking Lipid Fragments, a list of theoretical lipid fragments is displayed.

7. On clicking the fragment name, fragment structure is displayed.

8. You can export search results in HTML format. Go to File> Generate Report. A Generate

Report dialog is launched. Select lipid information to be exported.

SimLipid 3.5 27

The results can be exported in HTML file formats for sharing information with colleagues or

publishing results.

MS/MS Annotation

1. After performing lipid a search, switch to the MS/ MS Annotation tab. Theoretical lipid

fragments are matched against experimental product ions.

2. Matched m/z values are highlighted in blue alongwith the name of the lipid fragment.

Unmatched m/z values are highlighted in yellow.

3. Fragment Structure : On selecting a particular lipid fragment and clicking Fragment

Structure, the fragment structure is displayed.

4. Select All/Clear All: Select the rows manually from the MS/MS annotation table or use the

Select All button to select all the ions or Clear All to remove the selection.

Note: If more than one fragment matches a given m/z value then the fragment annotation is

displayed in a separate row using decimal values. For Eg 1.1, 1.2 etc.

SimLipid 3.5 28

Export Annotation

You can export the MS/ MS Annotation table in HTML format.

1. Select the values to be exported manually or click Select All.

2. Click Export Annotation. Select the parameters to include in the HTML report.

Plot Mass Spectrum

SimLipid allows you to annotate the MS/MS spectrum with matched lipid fragments.

1. Select a lipid to be annotated from the MS/ MS Annotation tab

2. To annotate specific fragments on the spectrum, select the interested fragments and click Plot

Spectrum. Clicking Plot Spectrum without making a selection annotates all the fragments.

3. A mass spectrum is displayed depicting lipid fragments on the selected peaks in case you have

selected specific fragments. Else, matched fragments are annotated on the peaks.

4. The annotated mass spectra can be re-sized either to fit a page or based on the area of

interest. You can zoom in/out into a specific plot locations or specify m/z range to display specific

plot sections for better analysis of the annotated spectra. . You can search a particular lipid

fragment by entering its m/z or intensity value in the Fragment Information table and clicking the

Search button.

5. The mass spectra can be exported as image in PNG/JPEG file format.

Comprehensive Report of Lipids Identified using MS and Complementary MS/MS Data

This module allows users to generate a comprehensive report of the lipids identified from an MS

and its complementary MS/MS or MSE data in MS Excel/ CSV/ HTML format.

To generate a report of MS and its complementary MS/MS data:

1. Perform MS and MS/MS lipid search.

2. Select the peak list or scan corresponding to the MS data from the project management panel.

3. Go to File> Compare Results>MS & MS/ MS Data. A Compare MS and MS/ MS Data

Results dialog is launched.

..

SimLipid 3.5 29

4. Select MS/ MS: You can select MS/ MS profile from the drop down list. For MSE files, MS/ MS

profile is selected automatically by the program.

5. On clicking the i button, Profile name and its Precursor Ion m/z is displayed.

SimLipid 3.5 30

6. Lipid Options: Select Lipid Options for MS Search and MS/ MS Search to be included in

the report.

a) MS Search Options are Experimental m/z, Intensity, Charge State, Retention Time and Drift

Time.

b) MS/ MS Search Options are Theoretical m/z, Lipid Score, Category, Abbreviation/Common

Name, Chemical Composition, Lipid Structure, Lipid Rank up to, Export lipids with distinct

chemical composition for the same rank and #Carbon: Double Bonds.

c) Lipid Rank up to allows users to select lipids up to rank 5.

d) Export Lipids with Distinct Chemical Composition for the Same Rank: If a user has

checked this option, multiple lipid IDs (of the same rank) with the same Chemical Composition

are not exported. By default the first Lipid ID is exported.

SimLipid 3.5 31

7. Output Options for Export Report:

The results can be exported in MS Excel/ CSV/ HTML format for sharing information with

colleagues or publishing results.

Comparative Report to Validate Lipids Identified in MS or MS/MS Data from Variable Ion Modes

SimLipid can generate a comparative report to validate lipids identified in different ion

mode/adducts. Lipid species identified from MS or MS/ MS spectra in variable ion modes and

adducts are aligned on the basis of similar Lipid IDs observed on both the spectra and exported

in MS Excel/CSV/ HTML format.

Steps to combine results and create comparative report to validate multiple MS/ MS data

sets:

1. Perform MS or MS/ MS Search on two peak list data sets that you intend to compare. If a

search is already performed, select the peak list scan from the project management panel.

2. Go to File>Compare Results>Multiple Data Sets. A Combine Multiple Data Sets Results

dialog is launched.

.

3. Select Data Type: Select Data type, MS data or MS/MS data.

SimLipid 3.5 32

4. Select Profile: Select Profile 1 and Profile 2. Profile 1 and Profile 2 should be two distinct

profiles. Click Add to create a set, selected profiles used for the set are displayed under column 1

and column 2 of the table. In the third column, the MS level of the set is displayed.

5. Select the Lipid Options for MS or MS/MS Search to be included in report:

MS Search options: are m/z, Intensity, Ion Type, Abbreviation/Common Name, Delta Mass,

Category, Lipid Structure, Retention Time, Drift Time and #Carbon: Double Bonds. MS/MS

Search options: Experimental m./z, Theoretical m/z, Delta Mass, Lipid Rank, Lipid Score, Lipid

Structure and #Carbon:Double Bonds.

6. Output Options for Export Report:

The results can be exported in MS Excel/CSV/ HTML format for sharing information with


SimLipid 3.5 33

Compare Profile MS Spectra

This feature enables comparing profiled MS spectra facilitating comparison of the lipids identified

from different biological samples. A comprehensive report in MS Excel/ CSV/ HTML format can

also be generated.

To generate a report for Profiled MS data:

1. Select the profiled MS peak list/scan from the project management panel.

2. Go to File> Compare Results> Profiled MS Spectra.

3. Compare Profiled MS Spectra Results dialog is launched.

SimLipid 3.5 34

4. Select Profile: Select the profiled MS spectra with which to compare the results. Click Create

Set. 5

5. The profiles selected for comparison are displayed under the table below. Up to 5 profiles can

be compared.

6. Select the Lipid Options to be included in the report: MS Search options: m/z, Intensity,

Chemical Composition, # Carbon: Double Bond, Abbreviation/Common Name, Delta Mass,

Category, Lipid Structure, Mass Spectrum, Category, Retention Time and Drift Time.

Choose the Filtering Options:

a) m/z: All matched lipids corresponding to an m/z value will be included in the report. Multiple

lipids may be matched for an m/z value.

b) Distinct m/z: Only a single lipid species corresponding to an m/z value will be included in the

report.

c) Distinct Chemical Formula: Among all the lipids reported for a particular m/z value, those with

different different chemical formula will only be reported.

7. The comparative report can further be organized by sorting lipids on m/z, intensity, retention

time etc.

8. Output Options for Export Report: The results can be exported in MS Excel/CSV/ HTML

format for sharing information with colleagues or publishing results.

Compare MS/MS Results by Aligning Retention Time

This feature enables aligning MS/MS search results from different chromatographic runs based

on retention time facilitating comparison of the lipids identified using variable ion modes. A

comprehensive report of aligned MS/ MS spectra can also be generated in Excel/ CSV/ HTML

format. The report includes matched fragment ions and observed intensity values facilitating

validation of lipids using MS/MS fragmentation patterns in variable ion modes.

For example, a PC lipid can be validated by checking if the head group fragment ions are

observed in the positive ion mode spectrum and if the fatty acyl ions are observed in the

corresponding negative ion mode spectrum.

To align MS/MS results by Retention Time

1. Go to File> Compare Results> Align Retention Time.

2. Align Retention Time dialog is launched.

SimLipid 3.5 35

3. Select Profile1, Profile 2 and create a set. If the Retention Time option is not checked, then all

the scans present in the files are imported in the table below. You can specify a retention time

range to import scans within a specified time window. Click OK.

4. You can also specify a tolerance range between 0 – 2 for the retention time.

5. The scans are displayed in the table below.

6. Select the Lipid Options to be included in the report: Experimental m/z, Intensity, Delta Mass,

Chemical Composition, Lipid Rank, Lipid Score, # Carbon: Double Bond, Lipid Structure,

Matched Fragments.

SimLipid 3.5 36

7. Output Options for Export Report: The results can be exported in MS Excel/CSV/ HTML

format for publishing results to the lab notebook .

High Throughput Lipid Search

High throughput lipid search module of SimLipid enables lipid profiling using MS data and

subsequent structural elucidation of lipids using MS/MS and MSE data. Users can import 20000

scans or peak list data sets and analyze 1000 scans in batch mode.

For Agilent's CEF files, SimLipid can now import MS and MS/MS data from 100 CEF files in batch

mode in a single click.

User can also perform neutral mass search using the ion species as well as the isotope cluster

information already available in the files. The throughput of the batch search is increased to 1000

compound MS and MS/MS profiles from the previous 300 compound profiles.

For easy selection and analysis in batch mode, scans/peak list data sets are numbered. User can

specify the range for which batch search they need triggered. Users can trigger multiple batch

searches using the program's powerful multi-thread analysis.

High throughput search will have 3 options:

i) Agilent Data

ii) Waters MSE

Data

iii) Other Data

High Throughput Lipid Search for Agilent Data

1. Go to Analyze> High Throughput Lipid Search> Agilent Data or click from toolbar. A

profile selection dialog launches. Select profile(s) and click OK.

SimLipid 3.5 37

2. A High Throughput Lipid Search dialog launches.

3. All the profiles loaded in the project management panel are displayed in the table. Profiles to

be analyzed can be selected here by checking appropriate boxes or by specifying a range. For

the latter, click Specify Range, enter the serial numbers of the scans or peak list data sets for

analysis and click OK.

4. Specify the search parameters for HTP search and click Search. You can view the status of

HTP Search by View> High Throughput Search Status.

5. If Search based on specified ion species by ignoring all the information available in the

files option is unchecked, you can perform neutral mass search using the ion species as well as

the isotope cluster information already available in the files.

High Throughput Lipid Search for Waters MSE Data

1. Go to Analyze> High Throughput Lipid Search> Waters MSE

Data or click from

toolbar. A High Throughput Lipid Search dialog launches.

SimLipid 3.5 38





3. If the precursor m/z is not available for a scan, then the Precursor Ion m/z column is left blank.

Click Edit to specify the Precursor Ion and Charge State information.

SimLipid 3.5 39

4. Specify the search parameters for HTP search.

5. Click Search to trigger a high throughput search for the selected range of scans. A search ID is

generated for a search launched.

High Throughput Lipid Search for Other Data

1. Go to Analyze> High Throughput Lipid Search> Other Data or click from toolbar. A

profile selection dialog launches. Select profile(s) and click OK.

2. A High Throughput Lipid Search dialog launches.

SimLipid 3.5 40





3. If the precursor m/z is not available for a scan, then the Precursor Ion m/z column is left blank.

Click Edit to specify the Precursor Ion and Charge State information.

SimLipid 3.5 41

4. Specify the search parameters for HTP search.

5. Click Search to trigger a high throughput search for the selected range of scans. A search ID is

generated for a search launched.

View the status of HTP Search and Fetch the Results

1. You can view the status of HTP Search by View> High Throughput Search Status.

2. Fetching High Throughput Results: To view the results of high throughput search, go to View>

Load High Throughput Results>HTP Request ID. HTP report is loaded in selected file format

and saved at desired location.

3. Select a scan or peak list data set from the project management panel. Complete search result

of the scan is loaded. Theoretical fragments, annotated peak list and labeled spectrum can be

explored from here as well.

Generate High Throughput Report

You can export the search results in a report format that can be printed. You can select up to

1000 profiles that needs to be included in the report by checking the boxes of the corresponding

fields.

1. Choose File>Generate High Throughput Report. A profile selection dialog launches. Select

profile(s) and click OK.

SimLipid 3.5 42

SimLipid 3.5 43

2. A Generate High Throughput Report dialog is launched.

3. Select the MS or MS/ MS options to be exported and click OK.

4. The results can be exported in CSV/ XLS/ HTML file formats for sharing information with


Generate Report

You can export the search results in a report format that can be printed. You can select the data

that needs to be included in the report by checking the boxes of the corresponding fields.

1. Choose File>Generate Report.

2. A Generate Report dialog is launched.

SimLipid 3.5 44

It has three sections: Lipid Selection, Lipid Options and Output Options.

a) Lipid Selection: You can select the lipids of interest from the list in the result panel by

selecting the check boxes on the left side of each of the reported lipid. You can select all the

lipids at once by selecting the check box at the top left corner of the result panel with symbol #.

All the selected lipids are exported directly.

b) Lipid Options: It has labels for exporting Lipid information along with check box. You can

select the information to be exported. For example m/z, Intensity, Ions/Charge, Lipid

Abbreviation, Delta Mass, Lipid Structure and Mass Spectrum.

c) File Options: The results can be exported in HTML file formats for sharing information with


3. Click OK. Results are exported in the selected format.

Export Results

You can save the search results at any desired location by selecting the Export Results option.

The results are exported to csv/xls file format for further processing.

1. Go to File>Export Results.

SimLipid 3.5 45

2. An Export Results dialog is launched.

It has three sections: Lipid Selection, Lipid Options and Output Options.

a) Lipid Selection: You can select the lipids of interest from the list in the result panel by clicking

the check boxes against the reported lipids. You can select all the lipids at once by selecting the

check box at the top left corner of the results panel with symbol #. b) Lipid Options: You can

select the information to be exported along with the lipid ID and sequence. For example m/z,

Intensity, Ions/Charge, Lipid Abbreviation and Delta Mass.

c) File Options: The results can be exported in .xls or csv file format for sharing information with


3. Click OK. Results are exported in the selected format.

Export Results to Agilent's CEF File

You can write SimLipid search results to Agilent CEF files for downstream processing in

MassHunter and MPP.

1. Go to File> Export Results to CEF.

2. Select Profile dialog is launched which displays all the profiles for which lipid profiling has

been performed. You can select multiple profiles and specify the location of file to be exported.

3. SimLipid can export and write the lipid search results to 25 CEF files.

4. For a compound ID having multiple MS/ MS profiles with different ion species, e.g., M+H,

M+Na and M+K, the lipid ID with highest probability score is exported to the CEF file.

SimLipid 3.5 46

5. Lipids profiled using MS data, the lipid ID with lowest delta mass is exported to the CEF file.

Find Lipids

You can search a lipid structure of interest in the SimLipid database by lipid abbreviation/mass/

chemical composition/lipid ID.

1. Go to Tools>Find Lipids.

2. A Find Lipids dialog box will be launched. It has four options for searching:

a) Lipid Abbreviation

b) Lipid Mass

c) Lipid Composition

d) Lipid ID

a) Lipid Search with Lipid Abbreviation

1. Select Lipid Abbreviation as an option for lipid search.

2. Enter lipid abbreviation in the text field. The format of writing lipid abbreviation is Head

group(sn1/sn2). For example: PC(16:0/12:0), PS(16:0/18:1(9Z)). “Z” indicates the number of

double bonds at the 9th position.

3. You can filter search results by specifying Total No of Carbons and Total No of Double Bond.

4. Click Search. Resultant lipids are displayed in the Search Results tab.

SimLipid 3.5 47

b) Lipid Search with Lipid Mass

1. Select Lipid Mass as an option for lipid search.

2. Enter lipid mass in the text field.

3. You can filter search results by specifying Total No of Carbons and Total No of Double Bond.


c) Lipid Search with Lipid Composition

1. Select Lipid Composition as an option for lipid search

SimLipid 3.5 48

2. Enter lipid composition in the text field. For example: C39H76NO8P

3. Click Search. Resultant lipids are displayed in the Search Results tab

d) Lipid Search with Lipid ID

1. Select Lipid ID as an option for lipid search.

2. Enter lipid ID in the text field. For example: LMGP01010507


Lipid Quantitation

SimLipid enables relative quantitation of lipids corresponding to the observed peaks on MS

spectra. Quantification is done by normalizing the intensity values based on the observed

intensity of an internal standard. To enable quantification of lipids from low resolution spectra, the

program corrects the intensities for isotopic overlapping of peaks. This facilitates accurate

quantification of lipids from biological mixtures. The quantified data along with other information

such as peak m/z, molecular formula of lipids, corrected, uncorrected and normalized intensities

in different samples can be exported to a spreadsheet compatible .csv and .xls formats.

Steps to perform lipid quantitation:

1. Go to Analyze> Lipid Quantitation >Select MS Profile or click from the tool bar.

SimLipid 3.5 49

2. Select MS Profile dialog launches. "Select Profile" shows all the MS data files/scans available

in the project, choose MS Profile(s) and click the button >> to include in the list for which quantity

of lipids have to be measured.

3. You can select multiple MS data files using the Select All option from the drop down list. At a

time, you can select up to 10 MS scans.

4. Selected profiles are displayed under the Selected profiles/Selected Scans column. For .txt

files, Selected Profile is the same as the Selected Scans.

5. Data Pre-processing Options: There are two options for data pre-processing.

a) m/z Options:

Here, you can perform the following pre-processing steps:

i) Consider m/z peaks observed in all the samples: Only those m/z values which are observed

in every MS data file/scan are considered. If it is left unchecked, then a union of m/z values

observed in all the selected MS data files is considered.

Note: If an m/z is not observed in the sample, its intensity is considered as zero.

ii) m/z precision (decimal points): You can specify the accuracy the program should use for its

SimLipid 3.5 50

calculations. By default, it is set as 1 i.e., precision up to first decimal value. You can set any

value between 0 and 4.

Example: m/z precision (decimal points): 0

If 701.5 is the observed m/z value, then SimLipid rounds it up to 702.

b) Intensity Options:

Here, you can perform the following pre-processing steps:

i) Multiple peaks with same m/z within round off limits:

You can either select the Average Intensity or Highest Intensity option.

If Average Intensity is selected, SimLipid calculates the average intensity from multiple peaks

which have the same m/z values (after rounding them off).

If Highest Intensity is selected, SimLipid searches for the highest intensity from multiple peaks

which have the same m/z values (after rounding them off).

ii) Threshold Intensity:

For Threshold Intensity, you can select either Relative or Value. SimLipid ignores all the peaks

below the specified threshold.

6. Processed data is loaded in MS Profile tab.

7. After pre-processing peak lists, go to Analyze> Lipid Quantitation>Search Lipids or click

from the tool bar.

SimLipid 3.5 51

8. A Search Lipids dialog launches. You can specify Error Tolerance, Polarity, Lipid Category

and Lipid Class.

9. On clicking Search, the analysis results are displayed in the Search Results tab.

10. You can also select to view the distinct chemical formula or m/z value for a reported peak.

11. To perform Lipid Quantitation, you need to mark a peak m/z as an Internal Standard by

clicking Mark/ Unmark Internal Standard.

a) When an m/z value that matches a lipid is selected as an Internal Standard, the Name of and

Amount (µg/mol) of the internal standard must be specified.

b) When an m/z value that does not match any lipid is selected as an Internal Standard, the

Name, Lipid Category, Lipid Class, Chemical Formula, Amount(µg/mol) and Total No. of Double

Bonds must be specified.

13. Only one Internal Standard can be selected for a lipid class.

14. For low resolution data, you can perform Isotopic Peak Correction to remove isotope

overlapping of peaks on the spectra, check Peak Correction and Click Generate Peak List

button.

SimLipid 3.5 52

15. If Peak Correction option is selected, Peak Correction dialog launches. You can calculate

isotope percentage for identified lipids either on the basis of the Parent Molecular Formula or

Neutral Loss Fragment. Enter the molecular formula of the neutral loss fragment which should

be subtracted from the parent molecular formula before calculating isotope percentage.Click

Calculate.

16. You will be directed to the Peak Lists tab. It displays a list of profiled lipids corresponding to

observed peaks in the spectra. The peak lists can be exported either in .csv, .xls or .html files by

clicking Export.

17. On clicking the Quantitation button, the intensity values of all the profiled lipids

corresponding to the peaks observed on the spectra are normalized based on the observed

intensity of the internal standard. Relative quantity of each lipid is calculated using the amount of

the internal standard. You will be directed to the Quantitation tab.

18. The Quantitation tab displays the Scan Name, Sample Name, Abbreviation /Common Name,

Lipid Class, Experimental m/z, Internal Standard, Uncorrected Intensity, Amount of Internal

Standard, Normalized Intensity and Relative Quantity.

SimLipid 3.5 53

19. On clicking Export, an Export Results dialog is launched. You can select the sample results

to be exported.

20. Reports can be organized by displaying results Sample wise or as Combined Samples.

Additionally, the results can be sorted either on Experimental m/z or on Lipid Class.

21. Select the lipid information to be exported. Search results can be exported in .csv as well as

in .xls format.

22. Click Plot Spectrum button. A Mass Spectrum displaying the low energy data is plotted.

System Specifications

Appendix Supported Operating System: Microsoft's Windows XP Professional\Vista\7.

System Requirements

SimLipid 3.5 54

For windows Required Recommended

CPU Pentium-III 800 MHz Pentium-IV 2.6 GHz

RAM 2 GB of available RAM 4 GB of available RAM

Hard Disk Drive Space 200 MB of available hard disk 500 MB of available hard disk

Screen Resolution 1024 x 768 > 1024 x 768

Copyright

Copyright © 2012 by PREMIER Biosoft International. All Rights Reserved.

Information in this manual may change without notice and does not represent a commitment on

the part of PREMIER Biosoft International.

The software described in this manual is provided by PREMIER Biosoft International under a

License Agreement. The software may be used only in accordance with the terms of the

agreement.

PREMIER Biosoft International (Premier) claims copyright to this program and documentation as

an unpublished work. Claim of copyright does not imply waiver of Premier's other rights.

This program and documentation are confidential and the property of Premier. Use, examination,

reproduction, copying, decompilation, transfer, and/or disclosure to others are strictly prohibited

except by express written agreement with Premier.

For more information on SimLipid and product updates, visit the PREMIER Biosoft web site at

http://www.PremierBiosoft.com

References

1) Hsu F.F. and Turk J (2000), "Characterization of phosphatidylinositol, phosphatidylinositol-4-

phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass

spectrometry: A mechanistic study", J. Am. Soc. Mass Spectrom., 11, 986-999.

SimLipid 3.5 55

2) Hsu,F.F. and Turk,J. ( 2000), "Charge-remote and charge-driven fragmentation processes in

diacyl glycerophosphoethanolamine upon low-energy collisional activation: A mechanistic

proposal", J. Am. Soc. Mass Spectrom., 11, 892-899.

3) Hsu,F.F. and Turk,J. (2000), "Characterization of phosphatidylethanolamine as a lithiated

adduct by triple quadrupole tandem mass spectrometry with electrospray ionization", J. Mass

Spectrom., 35, 596-606.

4) Hsu,F.F. and Turk J. (2000), "Charge-driven fragmentation processes in diacyl

glycerophosphatidic acids upon low-energy collisional activation. A mechanistic proposal", J. Am.

Soc. Mass Spectrom., 11, 797-803 .

5) Murphy Robert C. (2002), "Mass spectrometry of phospholipids: tables of molecular and

product ions", Illuminati Press, 71 s. ISBN: 0970283415.

6) Taguchi,R., Hayakawa,J., Takeuchi,Y. and Ishida,M. (2000), "Two-dimensional analysis of

phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry", J.

Mass Spectrom., 35, 953-966.

7) Zhu,J. and Cole, R.B. (2000), "Formation and decompositions of chloride adduct ions, [M + Cl]-

, in negative ion electrospray ionization mass spectrometry", J. Am. Soc. Mass Spectrom., 11,

932-941.

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