1

MRMPROBS tutorial

Edited in 20161116

Introduction

MRMPROBS is launched as a universal program for targeted metabolomics using not only multiple

reaction monitoring (MRM)- or selected reaction monitoring (SRM) but also SCAN and data

independent MSMS acquisition (DIA) data Originally the previous MRMPROBS program was

developed to deal with large scale MRM assayrsquos data sets monitoring 500-1000 small molecules in a

single run simultaneously The program provided 1) a user-friendly graphical user interface (GUI) for

data curation and 2) an objective evaluation system of small molecule identifications Here it was

expanded for DIA-MS data (like SWATH-MS) and for SCAN data (like GCMS and LCMS)

All data-processing workflow from data import to statistical analysis is supported This

tutorial will introduce the workflow for 1) MRM data 2) SWATH-MS (DIA) data and 3) GCMS

data for targeted metabolomics In this MRMPROBS project your feedback would be appreciated to

improve the identification and quantification systems as well as the user interface

Hiroshi Tsugawa

RIKEN Center for Sustainable Resource Science

hiroshitsugawarikenjp

MRMPROBS screenshot

2

Table of contents

Software environments 4

Required software programs and files 5

Project type and condition 6

ABF file conversion 7

Downloading the ABF converter 7

Check the conditions for file conversion 8

File conversion 10

Reference file format 11

Reference library for Project type 1 MRMPROBS key index = metabolite name (abf) 11

Reference library for Project type 2 MRMPROBS key index = Function (mzML) 14

Reference library for Project type 3 MRMPROBS key index = SCAN or DIA-MS (abf) 16

Reference format for DIA-MS data 16

Dictionary file for DIA-MS data processing 18

Reference format for GCMS and LCMS data 19

Starting MRMPROBS 20

Summary for MRM demonstration data sets 20

Starting up your project 21

Importing Abf files 22

Parameter 23

MRMPROBS viewer 25

Mouse operation in the chromatogram viewer 25

Library editor [optional] 27

Tool button 28

Tab 29

Button 30

List Box 31

Details on the MRMPROBS function 32

File menu 32

Data reprocessing 33

Statistical analysis 34

Missing value methods 35

Normalization 36

Window menu 38

View menu 39

Option menu 40

Export menu 41

3

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file 42

Appendix B Third option of MRMPROBS via mzML file 46

4

Software environments

Microsoft Windows 7 or later

NET Framework 40 or later

5

Required software programs and files

Reifycs Analysis Base File Converter (ABF file converter)

Download link httpwwwreifycscomAbfConverterindexhtml

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Demo files and the reference library (tab-delimited text file)

Example httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

MRMPROBS can import Analysis Base Framework (ABF) format data MRMPROBS extracts

chromatogram data together with the reference library including the name of the target metabolite its

retention-time and amplitude information and precursor mz and product mz The supported formats

for ABF conversion are Shimadzu Inc (LCD) Agilent Technologies (D) AB Sciex (WIFF) Waters

(RAW) and Thermo Fisher Scientific (RAW) MRMPROBS is also acceptable to a common data

format mzML converted by an open source file translator ProteoWizard The information is

described in Appendix B

3

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file 42

Appendix B Third option of MRMPROBS via mzML file 46

4

Software environments

Microsoft Windows 7 or later

NET Framework 40 or later

5

Required software programs and files

Reifycs Analysis Base File Converter (ABF file converter)

Download link httpwwwreifycscomAbfConverterindexhtml

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Demo files and the reference library (tab-delimited text file)

Example httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

MRMPROBS can import Analysis Base Framework (ABF) format data MRMPROBS extracts

chromatogram data together with the reference library including the name of the target metabolite its

retention-time and amplitude information and precursor mz and product mz The supported formats

for ABF conversion are Shimadzu Inc (LCD) Agilent Technologies (D) AB Sciex (WIFF) Waters

(RAW) and Thermo Fisher Scientific (RAW) MRMPROBS is also acceptable to a common data

format mzML converted by an open source file translator ProteoWizard The information is

described in Appendix B

4

Software environments

Microsoft Windows 7 or later

NET Framework 40 or later

5

Required software programs and files

Reifycs Analysis Base File Converter (ABF file converter)

Download link httpwwwreifycscomAbfConverterindexhtml

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Demo files and the reference library (tab-delimited text file)

Example httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

MRMPROBS can import Analysis Base Framework (ABF) format data MRMPROBS extracts

chromatogram data together with the reference library including the name of the target metabolite its

retention-time and amplitude information and precursor mz and product mz The supported formats

for ABF conversion are Shimadzu Inc (LCD) Agilent Technologies (D) AB Sciex (WIFF) Waters

(RAW) and Thermo Fisher Scientific (RAW) MRMPROBS is also acceptable to a common data

format mzML converted by an open source file translator ProteoWizard The information is

described in Appendix B

5

Required software programs and files

Reifycs Analysis Base File Converter (ABF file converter)

Download link httpwwwreifycscomAbfConverterindexhtml

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Demo files and the reference library (tab-delimited text file)

Example httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

MRMPROBS can import Analysis Base Framework (ABF) format data MRMPROBS extracts

chromatogram data together with the reference library including the name of the target metabolite its

retention-time and amplitude information and precursor mz and product mz The supported formats

for ABF conversion are Shimadzu Inc (LCD) Agilent Technologies (D) AB Sciex (WIFF) Waters

(RAW) and Thermo Fisher Scientific (RAW) MRMPROBS is also acceptable to a common data

format mzML converted by an open source file translator ProteoWizard The information is

described in Appendix B

6

Project type and condition

1 MRMPROBS key index = metabolite name (abf)

Use half-width alphanumeric symbols for compound names in MRM method setting

Do not use the same compound name at different transition (precursor-product) sets

2 MRMPROBS key index = Function (mzML)

lsquoFunction IDrsquo is utilized to extract the chromatogram data Users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format (see Appendix B)

The above two projects are for MRM data sets

3 MRMPROBS key index = SCAN or DIA-MS (abf)

MRMPROBS can import GCMS LCMS and LC-DIA-MS (like SWATH) data sets

Prepare the reference library (see Appendix C) to extract the certain retention time range and mz

values

Use half-width alphanumeric symbols for compound names

4 MRM-DIFF (abf mzML)

See httpprimepscrikenjpMetabolomics_SoftwareMRM-DIFFindexhtml

7

ABF file conversion

Downloading the ABF converter

1 Go to httpwwwreifycscomAbfConverterindexhtml

2 Check the requirements and license terms and download the converter

File converter is freely available

8

Check the conditions for file conversion

To convert files of some MS vendors including Bruker LECO Shimadzu Thermo and Waters the

specific data access library needs to be installed on your PC

Also see FAQ for ABF converter

httpprimepscrikenjpMetabolomics_SoftwareMS-DIALindex3html

Summary of PC condition required for file conversion

Vendor Formats Required

Agilent D None but the files from Chemstation should be

converted to netCDF

Bruker D CompassXtract

LECO PEG All PEG files should be first converted to netCDF

(AIA)

Sciex WIFF None

Shimadzu for

GCMS QGD GCMS solution

Shimadzu for

LCMS LCD LCMS solutions

Thermo RAW MSFileReader

Waters RAW MassLynx Raw Data Reader Interface Library

netCDF CDF Microsoft Visual J 20

FAQ

Agilent Almost all of Agilentrsquos raw data (D) can readily be converted without any additional

steps However the only exception is GCMS data sets obtained from ChemStation which

cannot be directly converted to ABF Therefore you have to convert your files into netCDF

(AIA) in ChemStation Then convert your AIA files into ABF using our file converter

Bruker Go to

httpswwwbrukercomservicesupport-upgradessoftware-downloadsmass-spectrometryhtml

You have to register first and obtain the installer Finally download and install the version

compatible with your operating system environment (32-bit or 64-bit)

LECO All of your data has to be converted to netCDF (AIA) first Then convert them into ABF

using our file converter

Shimadzu The compound table of the lcd file must include the MRM conditions of the ldquolcmrdquo

file How to set this is described in Appendix A-1 In case that you had a direct conversion of

raw data please convert your data into the common data format (netCDF or mzML) For

GCMS convert your data into netCDF using GCMS solution For LC-ITTOFMS convert your

8

Check the conditions for file conversion

To convert files of some MS vendors including Bruker LECO Shimadzu Thermo and Waters the

specific data access library needs to be installed on your PC

Also see FAQ for ABF converter

httpprimepscrikenjpMetabolomics_SoftwareMS-DIALindex3html

Summary of PC condition required for file conversion

Vendor Formats Required

Agilent D None but the files from Chemstation should be

converted to netCDF

Bruker D CompassXtract

LECO PEG All PEG files should be first converted to netCDF

(AIA)

Sciex WIFF None

Shimadzu for

GCMS QGD GCMS solution

Shimadzu for

LCMS LCD LCMS solutions

Thermo RAW MSFileReader

Waters RAW MassLynx Raw Data Reader Interface Library

netCDF CDF Microsoft Visual J 20

FAQ

Agilent Almost all of Agilentrsquos raw data (D) can readily be converted without any additional

steps However the only exception is GCMS data sets obtained from ChemStation which

cannot be directly converted to ABF Therefore you have to convert your files into netCDF

(AIA) in ChemStation Then convert your AIA files into ABF using our file converter

Bruker Go to

httpswwwbrukercomservicesupport-upgradessoftware-downloadsmass-spectrometryhtml

You have to register first and obtain the installer Finally download and install the version

compatible with your operating system environment (32-bit or 64-bit)

LECO All of your data has to be converted to netCDF (AIA) first Then convert them into ABF

using our file converter

Shimadzu The compound table of the lcd file must include the MRM conditions of the ldquolcmrdquo

file How to set this is described in Appendix A-1 In case that you had a direct conversion of

raw data please convert your data into the common data format (netCDF or mzML) For

GCMS convert your data into netCDF using GCMS solution For LC-ITTOFMS convert your

9

data into mzML using ProteoWizard (httpproteowizardsourceforgenetindexshtml) Then

convert them into ABF

Thermo The following link explains how to install MSFileReader

httpfieldsscrippsedurawconv For GCMS data you may have to convert your data into

netCDF Then convert them into ABF using our converter We validated the direct conversion of

GC-QExactive raw data to ABF but some GCMS data (DSQ etc) had to be converted to

netCDF first

Waters 1 Download MassLyncs Raw Data Reader Interface Library

(httpwwwwaterscomwaterssupportListhtmcid=511442ampfilter=documenttype|DWNLamploc

ale=en_US) 2 Unzip the archive file `watersrawsdkredistzip` and copy `MassLynxRawdll`

(64-bit) to `ABFCvtSvrWtrRw` folder in the ABF converter Note that 32ndashbit environments are

not supported yet for file conversion

NetCDF When you get an error about the J dependency problem download and install the

Microsoft Visual J 20 library at

httpswwwmicrosoftcomen-usdownloaddetailsaspxid=15468

10

File conversion

1 Start ldquoAnalysisBaseFileConverterexerdquo

2 Drag amp drop MS vendor files into this program

3 Click ldquoConvertrdquo

4 The ABF files are generated in the same directory as the raw data files

11

Reference file format

Reference library for Project type 1 MRMPROBS key index = metabolite name (abf)

Five items are required as tab-delimited format The header names are flexible but the item order

should be kept

1 column Compound namea

2 column Precursor mz (accurate mz information is rounded into nominal mz information)

3 column Product mz

4 column Retention time [min]

5 column Amplitude ratios []b

Notes aWhen you choose project 1 of MRMPROBS the name must be identical to the compound name in

the instrument setting window The compound name MUST be written by half-width alphanumeric

symbols

bAbout the amplitude ratio format

lsquo100rsquo value is recognized as the quantitative ion for targeted compounds and the other values are

recognized as the diagnostic ions for identification of targeted compounds

12

The quantitative ion must be set to each compound record and the current program does not

accept multiple quantitative ions for one compound it means that lsquo100rsquo value can be used only

once The below is the examples for reference records

Example only one transition for one metabolite

Thymine 125 4205 558 100

Example multiple transitions for one metabolite

G6P 2589 9705 921 100

G6P 2589 7905 921 301

G6P 2589 19915 921 55

Again MRMPROBS does not quantify the metabolite by the total ion chromatogram Instead

one quantitative transition is used for peak quantification (we call it the ldquotargetrdquo transition)

Enter the ratio [] against 100 as the amplitude for the qualifier transition (or the confirmation

transition) This information is used for peak identification (we call it the ldquoqualifierrdquo transition)

Note 1 You can edit the reference library and update its information in MRMPROBS However an

empty value cannot be accepted when the library is imported If you do not know the suitable

retention time and amplitude information for the metabolites enter arbitrary values for the

metabolites

Note 2 Users do not have to include all metabolite information you entered in the MS instrument

13

Note 3 Sometimes the tab-delimited file exported from Microsoft Excel includes unexpected hidden

trailing columns These unexpected columns after the lsquoRatiorsquo column cannot be handled by

MRMPROBS You can inspect the exported file by selecting a few rows (see below) If there are

selected characters after the last column (Ratio) edit the file in Excel to delete these columns and

re-export it again

Good example (no unexpected column)

Bad example (there are unexpected columns)

14

Reference library for Project type 2 MRMPROBS key index = Function (mzML)

Six items are required as tab-delimited text format The header names are flexible but the item order

should be followed (Here in order easily to see the library the reference was described in the

Microsoft Excel)

1 column Compound namea

2 column Function IDb

3 column Precursor mz (accurate mz information is rounded into nominal mz information)

4 column Product mz

5 column Retention time [min]

6 column Amplitude ratios []c

Notes aWhen you use project 2 of MRMPROBS the name doesnrsquot have to be identical to the compound

name in the instrument setting The compound name MUST be written by half-width alphanumeric

symbols

bThe function ID is the most important ID to use this option In the mzML data there is a markup

indicating a lsquoFunction IDrsquo which is unambiguous key to contact to the specific MRM chromatogram

for the retention time range the precursor ion and product ion In order to easily to see the

relationship between the function ID and the MRM information use the SeeMS program which can

be downloaded at ProteoWizard webpage httpproteowizardsourceforgenet

1 Open SeeMS

15

2 Select a mzML file

To find the identical function ID in your data use the Microsoft Excel sorting function and your

experiment condition file In the most of case the proteowizard is sorting the functions following the

order to 1 Precursor Ion 2 Product Ion 3 Retention time starting point

cAbout the amplitude ratio format

See the section of Reference library for Project type 1 MRMPROBS key index = metabolite name

(abf)

16

Reference library for Project type 3 MRMPROBS key index = SCAN or DIA-MS (abf)

Reference format for DIA-MS data

Users can utilize MRMPROBS software for scan type data such as GCMS LCMS and LC-data

independent MSMS (DIA-MS) The below figure is the reference library for DIA-MS data Here

our objective is to utilized DIA-MS data as MRM (what we call DIA-MRM for example

SWATH-MRM for SCIEX machine) This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

1 column Compound name

2 column Precursor mz

3 column Product mz

4 column Retention time [min]

5 column Amplitude ratios []

6 column RT begin start time to draw the chromatogram

7 column RT end end time to draw the chromatogram

8 column MS1 tolerance mass accuracy for survey scan MS data

17

9 column MS2 tolerance mass accuracy for MSMS spectra

10 column MS level put 1 for survey scan MS data (MS1) and put 2 for MSMS

11 column Class itrsquos used for the MRMPROBS viewer to filter out the chromatograms Set

lsquoNArsquo or something if not interest

Below is the description of the lsquobridgersquo from MS-DIAL to MRMPROBS

18

Dictionary file for DIA-MS data processing

The dictionary file should contain MS1 scan range and precursor window in combination with its

experimental ID

In the case of SWATH data-independent analysis the experiment file can be made at PeakView

(Show-gtsample information) Do not change the column orders The word ldquoSCANrdquo should be kept

19

Reference format for GCMS and LCMS data

MRMPROBS is improved to utilize single MS data such as GCMS and LCMS and the below

figure is the reference library for GCMS data The trick to import the single MS data sets is 1) to

assign the same values for product mz and MS2 tolerance as precursor mz and MS1 tolerance

respectively and 2) to assign lsquo1rsquo as MS level for all queries

This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

11

Reference file format

Reference library for Project type 1 MRMPROBS key index = metabolite name (abf)

Five items are required as tab-delimited format The header names are flexible but the item order

should be kept

1 column Compound namea

2 column Precursor mz (accurate mz information is rounded into nominal mz information)

3 column Product mz

4 column Retention time [min]

5 column Amplitude ratios []b

Notes aWhen you choose project 1 of MRMPROBS the name must be identical to the compound name in

the instrument setting window The compound name MUST be written by half-width alphanumeric

symbols

bAbout the amplitude ratio format

lsquo100rsquo value is recognized as the quantitative ion for targeted compounds and the other values are

recognized as the diagnostic ions for identification of targeted compounds

12

The quantitative ion must be set to each compound record and the current program does not

accept multiple quantitative ions for one compound it means that lsquo100rsquo value can be used only

once The below is the examples for reference records

Example only one transition for one metabolite

Thymine 125 4205 558 100

Example multiple transitions for one metabolite

G6P 2589 9705 921 100

G6P 2589 7905 921 301

G6P 2589 19915 921 55

Again MRMPROBS does not quantify the metabolite by the total ion chromatogram Instead

one quantitative transition is used for peak quantification (we call it the ldquotargetrdquo transition)

Enter the ratio [] against 100 as the amplitude for the qualifier transition (or the confirmation

transition) This information is used for peak identification (we call it the ldquoqualifierrdquo transition)

Note 1 You can edit the reference library and update its information in MRMPROBS However an

empty value cannot be accepted when the library is imported If you do not know the suitable

retention time and amplitude information for the metabolites enter arbitrary values for the

metabolites

Note 2 Users do not have to include all metabolite information you entered in the MS instrument

13

Note 3 Sometimes the tab-delimited file exported from Microsoft Excel includes unexpected hidden

trailing columns These unexpected columns after the lsquoRatiorsquo column cannot be handled by

MRMPROBS You can inspect the exported file by selecting a few rows (see below) If there are

selected characters after the last column (Ratio) edit the file in Excel to delete these columns and

re-export it again

Good example (no unexpected column)

Bad example (there are unexpected columns)

14

Reference library for Project type 2 MRMPROBS key index = Function (mzML)

Six items are required as tab-delimited text format The header names are flexible but the item order

should be followed (Here in order easily to see the library the reference was described in the

Microsoft Excel)

1 column Compound namea

2 column Function IDb

3 column Precursor mz (accurate mz information is rounded into nominal mz information)

4 column Product mz

5 column Retention time [min]

6 column Amplitude ratios []c

Notes aWhen you use project 2 of MRMPROBS the name doesnrsquot have to be identical to the compound

name in the instrument setting The compound name MUST be written by half-width alphanumeric

symbols

bThe function ID is the most important ID to use this option In the mzML data there is a markup

indicating a lsquoFunction IDrsquo which is unambiguous key to contact to the specific MRM chromatogram

for the retention time range the precursor ion and product ion In order to easily to see the

relationship between the function ID and the MRM information use the SeeMS program which can

be downloaded at ProteoWizard webpage httpproteowizardsourceforgenet

1 Open SeeMS

15

2 Select a mzML file

To find the identical function ID in your data use the Microsoft Excel sorting function and your

experiment condition file In the most of case the proteowizard is sorting the functions following the

order to 1 Precursor Ion 2 Product Ion 3 Retention time starting point

cAbout the amplitude ratio format

See the section of Reference library for Project type 1 MRMPROBS key index = metabolite name

(abf)

16

Reference library for Project type 3 MRMPROBS key index = SCAN or DIA-MS (abf)

Reference format for DIA-MS data

Users can utilize MRMPROBS software for scan type data such as GCMS LCMS and LC-data

independent MSMS (DIA-MS) The below figure is the reference library for DIA-MS data Here

our objective is to utilized DIA-MS data as MRM (what we call DIA-MRM for example

SWATH-MRM for SCIEX machine) This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

1 column Compound name

2 column Precursor mz

3 column Product mz

4 column Retention time [min]

5 column Amplitude ratios []

6 column RT begin start time to draw the chromatogram

7 column RT end end time to draw the chromatogram

8 column MS1 tolerance mass accuracy for survey scan MS data

17

9 column MS2 tolerance mass accuracy for MSMS spectra

10 column MS level put 1 for survey scan MS data (MS1) and put 2 for MSMS

11 column Class itrsquos used for the MRMPROBS viewer to filter out the chromatograms Set

lsquoNArsquo or something if not interest

Below is the description of the lsquobridgersquo from MS-DIAL to MRMPROBS

18

Dictionary file for DIA-MS data processing

The dictionary file should contain MS1 scan range and precursor window in combination with its

experimental ID

In the case of SWATH data-independent analysis the experiment file can be made at PeakView

(Show-gtsample information) Do not change the column orders The word ldquoSCANrdquo should be kept

19

Reference format for GCMS and LCMS data

MRMPROBS is improved to utilize single MS data such as GCMS and LCMS and the below

figure is the reference library for GCMS data The trick to import the single MS data sets is 1) to

assign the same values for product mz and MS2 tolerance as precursor mz and MS1 tolerance

respectively and 2) to assign lsquo1rsquo as MS level for all queries

This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

12

The quantitative ion must be set to each compound record and the current program does not

accept multiple quantitative ions for one compound it means that lsquo100rsquo value can be used only

once The below is the examples for reference records

Example only one transition for one metabolite

Thymine 125 4205 558 100

Example multiple transitions for one metabolite

G6P 2589 9705 921 100

G6P 2589 7905 921 301

G6P 2589 19915 921 55

Again MRMPROBS does not quantify the metabolite by the total ion chromatogram Instead

one quantitative transition is used for peak quantification (we call it the ldquotargetrdquo transition)

Enter the ratio [] against 100 as the amplitude for the qualifier transition (or the confirmation

transition) This information is used for peak identification (we call it the ldquoqualifierrdquo transition)

Note 1 You can edit the reference library and update its information in MRMPROBS However an

empty value cannot be accepted when the library is imported If you do not know the suitable

retention time and amplitude information for the metabolites enter arbitrary values for the

metabolites

Note 2 Users do not have to include all metabolite information you entered in the MS instrument

13

Note 3 Sometimes the tab-delimited file exported from Microsoft Excel includes unexpected hidden

trailing columns These unexpected columns after the lsquoRatiorsquo column cannot be handled by

MRMPROBS You can inspect the exported file by selecting a few rows (see below) If there are

selected characters after the last column (Ratio) edit the file in Excel to delete these columns and

re-export it again

Good example (no unexpected column)

Bad example (there are unexpected columns)

14

Reference library for Project type 2 MRMPROBS key index = Function (mzML)

Six items are required as tab-delimited text format The header names are flexible but the item order

should be followed (Here in order easily to see the library the reference was described in the

Microsoft Excel)

1 column Compound namea

2 column Function IDb

3 column Precursor mz (accurate mz information is rounded into nominal mz information)

4 column Product mz

5 column Retention time [min]

6 column Amplitude ratios []c

Notes aWhen you use project 2 of MRMPROBS the name doesnrsquot have to be identical to the compound

name in the instrument setting The compound name MUST be written by half-width alphanumeric

symbols

bThe function ID is the most important ID to use this option In the mzML data there is a markup

indicating a lsquoFunction IDrsquo which is unambiguous key to contact to the specific MRM chromatogram

for the retention time range the precursor ion and product ion In order to easily to see the

relationship between the function ID and the MRM information use the SeeMS program which can

be downloaded at ProteoWizard webpage httpproteowizardsourceforgenet

1 Open SeeMS

15

2 Select a mzML file

To find the identical function ID in your data use the Microsoft Excel sorting function and your

experiment condition file In the most of case the proteowizard is sorting the functions following the

order to 1 Precursor Ion 2 Product Ion 3 Retention time starting point

cAbout the amplitude ratio format

See the section of Reference library for Project type 1 MRMPROBS key index = metabolite name

(abf)

16

Reference library for Project type 3 MRMPROBS key index = SCAN or DIA-MS (abf)

Reference format for DIA-MS data

Users can utilize MRMPROBS software for scan type data such as GCMS LCMS and LC-data

independent MSMS (DIA-MS) The below figure is the reference library for DIA-MS data Here

our objective is to utilized DIA-MS data as MRM (what we call DIA-MRM for example

SWATH-MRM for SCIEX machine) This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

1 column Compound name

2 column Precursor mz

3 column Product mz

4 column Retention time [min]

5 column Amplitude ratios []

6 column RT begin start time to draw the chromatogram

7 column RT end end time to draw the chromatogram

8 column MS1 tolerance mass accuracy for survey scan MS data

17

9 column MS2 tolerance mass accuracy for MSMS spectra

10 column MS level put 1 for survey scan MS data (MS1) and put 2 for MSMS

11 column Class itrsquos used for the MRMPROBS viewer to filter out the chromatograms Set

lsquoNArsquo or something if not interest

Below is the description of the lsquobridgersquo from MS-DIAL to MRMPROBS

18

Dictionary file for DIA-MS data processing

The dictionary file should contain MS1 scan range and precursor window in combination with its

experimental ID

In the case of SWATH data-independent analysis the experiment file can be made at PeakView

(Show-gtsample information) Do not change the column orders The word ldquoSCANrdquo should be kept

19

Reference format for GCMS and LCMS data

MRMPROBS is improved to utilize single MS data such as GCMS and LCMS and the below

figure is the reference library for GCMS data The trick to import the single MS data sets is 1) to

assign the same values for product mz and MS2 tolerance as precursor mz and MS1 tolerance

respectively and 2) to assign lsquo1rsquo as MS level for all queries

This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

13

Note 3 Sometimes the tab-delimited file exported from Microsoft Excel includes unexpected hidden

trailing columns These unexpected columns after the lsquoRatiorsquo column cannot be handled by

MRMPROBS You can inspect the exported file by selecting a few rows (see below) If there are

selected characters after the last column (Ratio) edit the file in Excel to delete these columns and

re-export it again

Good example (no unexpected column)

Bad example (there are unexpected columns)

14

Reference library for Project type 2 MRMPROBS key index = Function (mzML)

Six items are required as tab-delimited text format The header names are flexible but the item order

should be followed (Here in order easily to see the library the reference was described in the

Microsoft Excel)

1 column Compound namea

2 column Function IDb

3 column Precursor mz (accurate mz information is rounded into nominal mz information)

4 column Product mz

5 column Retention time [min]

6 column Amplitude ratios []c

Notes aWhen you use project 2 of MRMPROBS the name doesnrsquot have to be identical to the compound

name in the instrument setting The compound name MUST be written by half-width alphanumeric

symbols

bThe function ID is the most important ID to use this option In the mzML data there is a markup

indicating a lsquoFunction IDrsquo which is unambiguous key to contact to the specific MRM chromatogram

for the retention time range the precursor ion and product ion In order to easily to see the

relationship between the function ID and the MRM information use the SeeMS program which can

be downloaded at ProteoWizard webpage httpproteowizardsourceforgenet

1 Open SeeMS

15

2 Select a mzML file

To find the identical function ID in your data use the Microsoft Excel sorting function and your

experiment condition file In the most of case the proteowizard is sorting the functions following the

order to 1 Precursor Ion 2 Product Ion 3 Retention time starting point

cAbout the amplitude ratio format

See the section of Reference library for Project type 1 MRMPROBS key index = metabolite name

(abf)

16

Reference library for Project type 3 MRMPROBS key index = SCAN or DIA-MS (abf)

Reference format for DIA-MS data

Users can utilize MRMPROBS software for scan type data such as GCMS LCMS and LC-data

independent MSMS (DIA-MS) The below figure is the reference library for DIA-MS data Here

our objective is to utilized DIA-MS data as MRM (what we call DIA-MRM for example

SWATH-MRM for SCIEX machine) This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

1 column Compound name

2 column Precursor mz

3 column Product mz

4 column Retention time [min]

5 column Amplitude ratios []

6 column RT begin start time to draw the chromatogram

7 column RT end end time to draw the chromatogram

8 column MS1 tolerance mass accuracy for survey scan MS data

17

9 column MS2 tolerance mass accuracy for MSMS spectra

10 column MS level put 1 for survey scan MS data (MS1) and put 2 for MSMS

11 column Class itrsquos used for the MRMPROBS viewer to filter out the chromatograms Set

lsquoNArsquo or something if not interest

Below is the description of the lsquobridgersquo from MS-DIAL to MRMPROBS

18

Dictionary file for DIA-MS data processing

The dictionary file should contain MS1 scan range and precursor window in combination with its

experimental ID

In the case of SWATH data-independent analysis the experiment file can be made at PeakView

(Show-gtsample information) Do not change the column orders The word ldquoSCANrdquo should be kept

19

Reference format for GCMS and LCMS data

MRMPROBS is improved to utilize single MS data such as GCMS and LCMS and the below

figure is the reference library for GCMS data The trick to import the single MS data sets is 1) to

assign the same values for product mz and MS2 tolerance as precursor mz and MS1 tolerance

respectively and 2) to assign lsquo1rsquo as MS level for all queries

This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

14

Reference library for Project type 2 MRMPROBS key index = Function (mzML)

Six items are required as tab-delimited text format The header names are flexible but the item order

should be followed (Here in order easily to see the library the reference was described in the

Microsoft Excel)

1 column Compound namea

2 column Function IDb

3 column Precursor mz (accurate mz information is rounded into nominal mz information)

4 column Product mz

5 column Retention time [min]

6 column Amplitude ratios []c

Notes aWhen you use project 2 of MRMPROBS the name doesnrsquot have to be identical to the compound

name in the instrument setting The compound name MUST be written by half-width alphanumeric

symbols

bThe function ID is the most important ID to use this option In the mzML data there is a markup

indicating a lsquoFunction IDrsquo which is unambiguous key to contact to the specific MRM chromatogram

for the retention time range the precursor ion and product ion In order to easily to see the

relationship between the function ID and the MRM information use the SeeMS program which can

be downloaded at ProteoWizard webpage httpproteowizardsourceforgenet

1 Open SeeMS

15

2 Select a mzML file

To find the identical function ID in your data use the Microsoft Excel sorting function and your

experiment condition file In the most of case the proteowizard is sorting the functions following the

order to 1 Precursor Ion 2 Product Ion 3 Retention time starting point

cAbout the amplitude ratio format

See the section of Reference library for Project type 1 MRMPROBS key index = metabolite name

(abf)

16

Reference library for Project type 3 MRMPROBS key index = SCAN or DIA-MS (abf)

Reference format for DIA-MS data

Users can utilize MRMPROBS software for scan type data such as GCMS LCMS and LC-data

independent MSMS (DIA-MS) The below figure is the reference library for DIA-MS data Here

our objective is to utilized DIA-MS data as MRM (what we call DIA-MRM for example

SWATH-MRM for SCIEX machine) This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

1 column Compound name

2 column Precursor mz

3 column Product mz

4 column Retention time [min]

5 column Amplitude ratios []

6 column RT begin start time to draw the chromatogram

7 column RT end end time to draw the chromatogram

8 column MS1 tolerance mass accuracy for survey scan MS data

17

9 column MS2 tolerance mass accuracy for MSMS spectra

10 column MS level put 1 for survey scan MS data (MS1) and put 2 for MSMS

11 column Class itrsquos used for the MRMPROBS viewer to filter out the chromatograms Set

lsquoNArsquo or something if not interest

Below is the description of the lsquobridgersquo from MS-DIAL to MRMPROBS

18

Dictionary file for DIA-MS data processing

The dictionary file should contain MS1 scan range and precursor window in combination with its

experimental ID

In the case of SWATH data-independent analysis the experiment file can be made at PeakView

(Show-gtsample information) Do not change the column orders The word ldquoSCANrdquo should be kept

19

Reference format for GCMS and LCMS data

MRMPROBS is improved to utilize single MS data such as GCMS and LCMS and the below

figure is the reference library for GCMS data The trick to import the single MS data sets is 1) to

assign the same values for product mz and MS2 tolerance as precursor mz and MS1 tolerance

respectively and 2) to assign lsquo1rsquo as MS level for all queries

This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

15

2 Select a mzML file

To find the identical function ID in your data use the Microsoft Excel sorting function and your

experiment condition file In the most of case the proteowizard is sorting the functions following the

order to 1 Precursor Ion 2 Product Ion 3 Retention time starting point

cAbout the amplitude ratio format

See the section of Reference library for Project type 1 MRMPROBS key index = metabolite name

(abf)

16

Reference library for Project type 3 MRMPROBS key index = SCAN or DIA-MS (abf)

Reference format for DIA-MS data

Users can utilize MRMPROBS software for scan type data such as GCMS LCMS and LC-data

independent MSMS (DIA-MS) The below figure is the reference library for DIA-MS data Here

our objective is to utilized DIA-MS data as MRM (what we call DIA-MRM for example

SWATH-MRM for SCIEX machine) This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

1 column Compound name

2 column Precursor mz

3 column Product mz

4 column Retention time [min]

5 column Amplitude ratios []

6 column RT begin start time to draw the chromatogram

7 column RT end end time to draw the chromatogram

8 column MS1 tolerance mass accuracy for survey scan MS data

17

9 column MS2 tolerance mass accuracy for MSMS spectra

10 column MS level put 1 for survey scan MS data (MS1) and put 2 for MSMS

11 column Class itrsquos used for the MRMPROBS viewer to filter out the chromatograms Set

lsquoNArsquo or something if not interest

Below is the description of the lsquobridgersquo from MS-DIAL to MRMPROBS

18

Dictionary file for DIA-MS data processing

The dictionary file should contain MS1 scan range and precursor window in combination with its

experimental ID

In the case of SWATH data-independent analysis the experiment file can be made at PeakView

(Show-gtsample information) Do not change the column orders The word ldquoSCANrdquo should be kept

19

Reference format for GCMS and LCMS data

MRMPROBS is improved to utilize single MS data such as GCMS and LCMS and the below

figure is the reference library for GCMS data The trick to import the single MS data sets is 1) to

assign the same values for product mz and MS2 tolerance as precursor mz and MS1 tolerance

respectively and 2) to assign lsquo1rsquo as MS level for all queries

This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

16

Reference library for Project type 3 MRMPROBS key index = SCAN or DIA-MS (abf)

Reference format for DIA-MS data

Users can utilize MRMPROBS software for scan type data such as GCMS LCMS and LC-data

independent MSMS (DIA-MS) The below figure is the reference library for DIA-MS data Here

our objective is to utilized DIA-MS data as MRM (what we call DIA-MRM for example

SWATH-MRM for SCIEX machine) This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

1 column Compound name

2 column Precursor mz

3 column Product mz

4 column Retention time [min]

5 column Amplitude ratios []

6 column RT begin start time to draw the chromatogram

7 column RT end end time to draw the chromatogram

8 column MS1 tolerance mass accuracy for survey scan MS data

17

9 column MS2 tolerance mass accuracy for MSMS spectra

10 column MS level put 1 for survey scan MS data (MS1) and put 2 for MSMS

11 column Class itrsquos used for the MRMPROBS viewer to filter out the chromatograms Set

lsquoNArsquo or something if not interest

Below is the description of the lsquobridgersquo from MS-DIAL to MRMPROBS

18

Dictionary file for DIA-MS data processing

The dictionary file should contain MS1 scan range and precursor window in combination with its

experimental ID

In the case of SWATH data-independent analysis the experiment file can be made at PeakView

(Show-gtsample information) Do not change the column orders The word ldquoSCANrdquo should be kept

19

Reference format for GCMS and LCMS data

MRMPROBS is improved to utilize single MS data such as GCMS and LCMS and the below

figure is the reference library for GCMS data The trick to import the single MS data sets is 1) to

assign the same values for product mz and MS2 tolerance as precursor mz and MS1 tolerance

respectively and 2) to assign lsquo1rsquo as MS level for all queries

This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

17

9 column MS2 tolerance mass accuracy for MSMS spectra

10 column MS level put 1 for survey scan MS data (MS1) and put 2 for MSMS

11 column Class itrsquos used for the MRMPROBS viewer to filter out the chromatograms Set

lsquoNArsquo or something if not interest

Below is the description of the lsquobridgersquo from MS-DIAL to MRMPROBS

18

Dictionary file for DIA-MS data processing

The dictionary file should contain MS1 scan range and precursor window in combination with its

experimental ID

In the case of SWATH data-independent analysis the experiment file can be made at PeakView

(Show-gtsample information) Do not change the column orders The word ldquoSCANrdquo should be kept

19

Reference format for GCMS and LCMS data

MRMPROBS is improved to utilize single MS data such as GCMS and LCMS and the below

figure is the reference library for GCMS data The trick to import the single MS data sets is 1) to

assign the same values for product mz and MS2 tolerance as precursor mz and MS1 tolerance

respectively and 2) to assign lsquo1rsquo as MS level for all queries

This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

18

Dictionary file for DIA-MS data processing

The dictionary file should contain MS1 scan range and precursor window in combination with its

experimental ID

In the case of SWATH data-independent analysis the experiment file can be made at PeakView

(Show-gtsample information) Do not change the column orders The word ldquoSCANrdquo should be kept

19

Reference format for GCMS and LCMS data

MRMPROBS is improved to utilize single MS data such as GCMS and LCMS and the below

figure is the reference library for GCMS data The trick to import the single MS data sets is 1) to

assign the same values for product mz and MS2 tolerance as precursor mz and MS1 tolerance

respectively and 2) to assign lsquo1rsquo as MS level for all queries

This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

19

Reference format for GCMS and LCMS data

MRMPROBS is improved to utilize single MS data such as GCMS and LCMS and the below

figure is the reference library for GCMS data The trick to import the single MS data sets is 1) to

assign the same values for product mz and MS2 tolerance as precursor mz and MS1 tolerance

respectively and 2) to assign lsquo1rsquo as MS level for all queries

This library can be easily exported by MS-DIAL software

httpprimepscrikenjpMetabolomics_SoftwareMS-DIAL

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

20

Starting MRMPROBS

Summary for MRM demonstration data sets

1 Starting up your project

2 Importing Abf files

3 Setting parameters

4 Running the software (1-2 min sample)

The tutorial uses 40 demonstration files and the reference library which are downloadable from the

above link The common measurement conditions of the demonstration files were as follows

Liquid chromatography total 25 min run per sample with CELI L-column2 ODC (150 mmtimes21 mm

3 m)

Mass spectrometer MRM method with negative ion mode

Target metabolite number 60

Total transitions 166

The detail of experimental conditions is downloadable at the MRM Database section (Ion-pair

LC-QqQMS)

httpprimepscrikenjpMetabolomics_SoftwareMrmDatabaseindexhtml

21

Starting up your project

1 File New project

2 Chose a project type (select the top one for this demonstration)

22

Importing Abf files

Note

We recommend that the project folder be made for each batch experiment In the

MRMPROBS project two folders (raw processed) and one file (mth) are generated They

should be included in the same directory

The file name should be entered in half-width alphanumeric symbols

Select the file type of each file from ldquoSamplerdquo ldquoStandardrdquo ldquoBlankrdquo and QCrdquo QC is

required for the LOESS-based normalization method

Class ID is used for the color labels

Analytical order and class ID can be changed after data processing

23

Parameter

Select lsquoExampleLibrarytxtrsquo and set the above parameters for this demonstration

Note

If you want to edit or update the retention time and amplitudes of metabolites in your

reference library from an example file such as a QC or standard mixture check ldquocreate new

library and choose the file name to which you want to refer In this demonstration

lsquo20_STD10uM_02rsquo is selected

[Recommended]

Peak detection

Smoothing method linear weighted moving average

Smoothing level 1-2

Minimum peak width 3-5

Minimum peak height 50-100

Peak identification

Retention time tolerance As long as the reverse phase or hydrophilic interaction

chromatography LC are used 01-02 min is recommended

Amplitude tolerance 15

24

Minimum posterior Decide the minimum probability for peak identification MRMPROBS

calculates a probability for a peak ie ldquoprobability of true target metabolite given the calculated

scoresrdquo The detected peak less than this criterion is recognized as a false peak The

recommended value is 50-70

Note The first data processing including file import peak detection and peak identification requires

5-20 seconds (depending on machine specifications) per file

25

MRMPROBS viewer

Mouse operation in the chromatogram viewer

Main window

View mode

① Chromatogram window drag holding left click chromatogram scroll drag holding right click

chromatogram zoom

1

3

4

2

26

② Detected window left double-click the reverse triangle change the true peak right

double-click anywhere un-checked detected peaks

③ Retention time window drag holding right click warping on retention time range

④ Intensity window drag holding right click warping on intensity range

Edit mode

① Left click and drag on the peak edge [red square] change the location of the peak edge

② Right click and drag detect new peak

27

Library editor [optional]

You can update the retention time and amplitude ratio of each metabolite by means of the QC

sample file or the standard mixture file You can also change the target transition for peak

quantification

The default identification is based on the highest peak in the EIC chromatogram If you need to

change the true peak double click the reverse triangle

If you change the target transition select the target MRM transition from the ldquoTarget MRMrdquo

combo box

The right-column information including the compound name target MRM RT and amplitude

ratios are generated by double-clicking the compound name in the list box on the left or by

re-selecting the true peak in the EIC chromatogram viewer

If the object metabolite is not detected click the ldquoViewrdquo button at the top left and switch to

ldquoEditrdquo mode In the ldquoEditrdquo mode hold the right mouse click and drag the peak area you want to

detect

If you want to change the retention time or amplitude information manually just type what you

want in the textbox and click the ldquoUpdaterdquo button on the top right

If you want to save the updated library click the ldquoSaverdquo button on the top left side

Lastly after editing the reference library click the ldquoFinishrdquo button on the top left side

Note The details and the operation method for chromatogram viewer are described later

28

Tool button

File start new project open existing project save as a project and save the project

Data processing for data re-processing per file per metabolite or in all data sets

Statistical analysis data normalization and statistical analysis

Window tile setting of chromatogram viewer

View sorting of chromatogram viewer

Option re-define class ID and analytical order choose the internal standard decide ldquoincluderdquo or

ldquoexcluderdquo data for statistical analysis

Identification non-meaningful in MRMPROBS project

Export The result is exported in tab-delimited text format

Help show version information

29

Tab

Chromatogram All data manipulation tasks are performed here

Raw data matrix The peak quantification value of each file and each metabolite is shown here

Processed data matrix The normalized value of each file and each metabolite is shown here

Statistical result The result of statistical analysis is shown here

Raw data matrix

30

Button

Reset Reset the display range of chromatograms

View If you push this ldquoViewrdquo button the chromatogram viewer is changed to ldquoEditrdquo mode In

the ldquoEditrdquo mode you can modify the peak edge and detect new peaks manually

None The properties of detected peaks are shown in this ComboBox You can confirm the total

score (probability) rt similarity area value and reference RT in the chromatogram viewer

Height You can set the quantification mode The default is set by peak height Instead you can

change it to area mode By using the ldquoAllrdquo option the quantification mode is reflected =

implemented in all files and all metabolites

Processed You can see raw chromatograms as well as smoothed chromatograms

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

22

Importing Abf files

Note

We recommend that the project folder be made for each batch experiment In the

MRMPROBS project two folders (raw processed) and one file (mth) are generated They

should be included in the same directory

The file name should be entered in half-width alphanumeric symbols

Select the file type of each file from ldquoSamplerdquo ldquoStandardrdquo ldquoBlankrdquo and QCrdquo QC is

required for the LOESS-based normalization method

Class ID is used for the color labels

Analytical order and class ID can be changed after data processing

23

Parameter

Select lsquoExampleLibrarytxtrsquo and set the above parameters for this demonstration

Note

If you want to edit or update the retention time and amplitudes of metabolites in your

reference library from an example file such as a QC or standard mixture check ldquocreate new

library and choose the file name to which you want to refer In this demonstration

lsquo20_STD10uM_02rsquo is selected

[Recommended]

Peak detection

Smoothing method linear weighted moving average

Smoothing level 1-2

Minimum peak width 3-5

Minimum peak height 50-100

Peak identification

Retention time tolerance As long as the reverse phase or hydrophilic interaction

chromatography LC are used 01-02 min is recommended

Amplitude tolerance 15

24

Minimum posterior Decide the minimum probability for peak identification MRMPROBS

calculates a probability for a peak ie ldquoprobability of true target metabolite given the calculated

scoresrdquo The detected peak less than this criterion is recognized as a false peak The

recommended value is 50-70

Note The first data processing including file import peak detection and peak identification requires

5-20 seconds (depending on machine specifications) per file

25

MRMPROBS viewer

Mouse operation in the chromatogram viewer

Main window

View mode

① Chromatogram window drag holding left click chromatogram scroll drag holding right click

chromatogram zoom

1

3

4

2

26

② Detected window left double-click the reverse triangle change the true peak right

double-click anywhere un-checked detected peaks

③ Retention time window drag holding right click warping on retention time range

④ Intensity window drag holding right click warping on intensity range

Edit mode

① Left click and drag on the peak edge [red square] change the location of the peak edge

② Right click and drag detect new peak

27

Library editor [optional]

You can update the retention time and amplitude ratio of each metabolite by means of the QC

sample file or the standard mixture file You can also change the target transition for peak

quantification

The default identification is based on the highest peak in the EIC chromatogram If you need to

change the true peak double click the reverse triangle

If you change the target transition select the target MRM transition from the ldquoTarget MRMrdquo

combo box

The right-column information including the compound name target MRM RT and amplitude

ratios are generated by double-clicking the compound name in the list box on the left or by

re-selecting the true peak in the EIC chromatogram viewer

If the object metabolite is not detected click the ldquoViewrdquo button at the top left and switch to

ldquoEditrdquo mode In the ldquoEditrdquo mode hold the right mouse click and drag the peak area you want to

detect

If you want to change the retention time or amplitude information manually just type what you

want in the textbox and click the ldquoUpdaterdquo button on the top right

If you want to save the updated library click the ldquoSaverdquo button on the top left side

Lastly after editing the reference library click the ldquoFinishrdquo button on the top left side

Note The details and the operation method for chromatogram viewer are described later

28

Tool button

File start new project open existing project save as a project and save the project

Data processing for data re-processing per file per metabolite or in all data sets

Statistical analysis data normalization and statistical analysis

Window tile setting of chromatogram viewer

View sorting of chromatogram viewer

Option re-define class ID and analytical order choose the internal standard decide ldquoincluderdquo or

ldquoexcluderdquo data for statistical analysis

Identification non-meaningful in MRMPROBS project

Export The result is exported in tab-delimited text format

Help show version information

29

Tab

Chromatogram All data manipulation tasks are performed here

Raw data matrix The peak quantification value of each file and each metabolite is shown here

Processed data matrix The normalized value of each file and each metabolite is shown here

Statistical result The result of statistical analysis is shown here

Raw data matrix

30

Button

Reset Reset the display range of chromatograms

View If you push this ldquoViewrdquo button the chromatogram viewer is changed to ldquoEditrdquo mode In

the ldquoEditrdquo mode you can modify the peak edge and detect new peaks manually

None The properties of detected peaks are shown in this ComboBox You can confirm the total

score (probability) rt similarity area value and reference RT in the chromatogram viewer

Height You can set the quantification mode The default is set by peak height Instead you can

change it to area mode By using the ldquoAllrdquo option the quantification mode is reflected =

implemented in all files and all metabolites

Processed You can see raw chromatograms as well as smoothed chromatograms

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

23

Parameter

Select lsquoExampleLibrarytxtrsquo and set the above parameters for this demonstration

Note

If you want to edit or update the retention time and amplitudes of metabolites in your

reference library from an example file such as a QC or standard mixture check ldquocreate new

library and choose the file name to which you want to refer In this demonstration

lsquo20_STD10uM_02rsquo is selected

[Recommended]

Peak detection

Smoothing method linear weighted moving average

Smoothing level 1-2

Minimum peak width 3-5

Minimum peak height 50-100

Peak identification

Retention time tolerance As long as the reverse phase or hydrophilic interaction

chromatography LC are used 01-02 min is recommended

Amplitude tolerance 15

24

Minimum posterior Decide the minimum probability for peak identification MRMPROBS

calculates a probability for a peak ie ldquoprobability of true target metabolite given the calculated

scoresrdquo The detected peak less than this criterion is recognized as a false peak The

recommended value is 50-70

Note The first data processing including file import peak detection and peak identification requires

5-20 seconds (depending on machine specifications) per file

25

MRMPROBS viewer

Mouse operation in the chromatogram viewer

Main window

View mode

① Chromatogram window drag holding left click chromatogram scroll drag holding right click

chromatogram zoom

1

3

4

2

26

② Detected window left double-click the reverse triangle change the true peak right

double-click anywhere un-checked detected peaks

③ Retention time window drag holding right click warping on retention time range

④ Intensity window drag holding right click warping on intensity range

Edit mode

① Left click and drag on the peak edge [red square] change the location of the peak edge

② Right click and drag detect new peak

27

Library editor [optional]

You can update the retention time and amplitude ratio of each metabolite by means of the QC

sample file or the standard mixture file You can also change the target transition for peak

quantification

The default identification is based on the highest peak in the EIC chromatogram If you need to

change the true peak double click the reverse triangle

If you change the target transition select the target MRM transition from the ldquoTarget MRMrdquo

combo box

The right-column information including the compound name target MRM RT and amplitude

ratios are generated by double-clicking the compound name in the list box on the left or by

re-selecting the true peak in the EIC chromatogram viewer

If the object metabolite is not detected click the ldquoViewrdquo button at the top left and switch to

ldquoEditrdquo mode In the ldquoEditrdquo mode hold the right mouse click and drag the peak area you want to

detect

If you want to change the retention time or amplitude information manually just type what you

want in the textbox and click the ldquoUpdaterdquo button on the top right

If you want to save the updated library click the ldquoSaverdquo button on the top left side

Lastly after editing the reference library click the ldquoFinishrdquo button on the top left side

Note The details and the operation method for chromatogram viewer are described later

28

Tool button

File start new project open existing project save as a project and save the project

Data processing for data re-processing per file per metabolite or in all data sets

Statistical analysis data normalization and statistical analysis

Window tile setting of chromatogram viewer

View sorting of chromatogram viewer

Option re-define class ID and analytical order choose the internal standard decide ldquoincluderdquo or

ldquoexcluderdquo data for statistical analysis

Identification non-meaningful in MRMPROBS project

Export The result is exported in tab-delimited text format

Help show version information

29

Tab

Chromatogram All data manipulation tasks are performed here

Raw data matrix The peak quantification value of each file and each metabolite is shown here

Processed data matrix The normalized value of each file and each metabolite is shown here

Statistical result The result of statistical analysis is shown here

Raw data matrix

30

Button

Reset Reset the display range of chromatograms

View If you push this ldquoViewrdquo button the chromatogram viewer is changed to ldquoEditrdquo mode In

the ldquoEditrdquo mode you can modify the peak edge and detect new peaks manually

None The properties of detected peaks are shown in this ComboBox You can confirm the total

score (probability) rt similarity area value and reference RT in the chromatogram viewer

Height You can set the quantification mode The default is set by peak height Instead you can

change it to area mode By using the ldquoAllrdquo option the quantification mode is reflected =

implemented in all files and all metabolites

Processed You can see raw chromatograms as well as smoothed chromatograms

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

24

Minimum posterior Decide the minimum probability for peak identification MRMPROBS

calculates a probability for a peak ie ldquoprobability of true target metabolite given the calculated

scoresrdquo The detected peak less than this criterion is recognized as a false peak The

recommended value is 50-70

Note The first data processing including file import peak detection and peak identification requires

5-20 seconds (depending on machine specifications) per file

25

MRMPROBS viewer

Mouse operation in the chromatogram viewer

Main window

View mode

① Chromatogram window drag holding left click chromatogram scroll drag holding right click

chromatogram zoom

1

3

4

2

26

② Detected window left double-click the reverse triangle change the true peak right

double-click anywhere un-checked detected peaks

③ Retention time window drag holding right click warping on retention time range

④ Intensity window drag holding right click warping on intensity range

Edit mode

① Left click and drag on the peak edge [red square] change the location of the peak edge

② Right click and drag detect new peak

27

Library editor [optional]

You can update the retention time and amplitude ratio of each metabolite by means of the QC

sample file or the standard mixture file You can also change the target transition for peak

quantification

The default identification is based on the highest peak in the EIC chromatogram If you need to

change the true peak double click the reverse triangle

If you change the target transition select the target MRM transition from the ldquoTarget MRMrdquo

combo box

The right-column information including the compound name target MRM RT and amplitude

ratios are generated by double-clicking the compound name in the list box on the left or by

re-selecting the true peak in the EIC chromatogram viewer

If the object metabolite is not detected click the ldquoViewrdquo button at the top left and switch to

ldquoEditrdquo mode In the ldquoEditrdquo mode hold the right mouse click and drag the peak area you want to

detect

If you want to change the retention time or amplitude information manually just type what you

want in the textbox and click the ldquoUpdaterdquo button on the top right

If you want to save the updated library click the ldquoSaverdquo button on the top left side

Lastly after editing the reference library click the ldquoFinishrdquo button on the top left side

Note The details and the operation method for chromatogram viewer are described later

28

Tool button

File start new project open existing project save as a project and save the project

Data processing for data re-processing per file per metabolite or in all data sets

Statistical analysis data normalization and statistical analysis

Window tile setting of chromatogram viewer

View sorting of chromatogram viewer

Option re-define class ID and analytical order choose the internal standard decide ldquoincluderdquo or

ldquoexcluderdquo data for statistical analysis

Identification non-meaningful in MRMPROBS project

Export The result is exported in tab-delimited text format

Help show version information

29

Tab

Chromatogram All data manipulation tasks are performed here

Raw data matrix The peak quantification value of each file and each metabolite is shown here

Processed data matrix The normalized value of each file and each metabolite is shown here

Statistical result The result of statistical analysis is shown here

Raw data matrix

30

Button

Reset Reset the display range of chromatograms

View If you push this ldquoViewrdquo button the chromatogram viewer is changed to ldquoEditrdquo mode In

the ldquoEditrdquo mode you can modify the peak edge and detect new peaks manually

None The properties of detected peaks are shown in this ComboBox You can confirm the total

score (probability) rt similarity area value and reference RT in the chromatogram viewer

Height You can set the quantification mode The default is set by peak height Instead you can

change it to area mode By using the ldquoAllrdquo option the quantification mode is reflected =

implemented in all files and all metabolites

Processed You can see raw chromatograms as well as smoothed chromatograms

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

25

MRMPROBS viewer

Mouse operation in the chromatogram viewer

Main window

View mode

① Chromatogram window drag holding left click chromatogram scroll drag holding right click

chromatogram zoom

1

3

4

2

26

② Detected window left double-click the reverse triangle change the true peak right

double-click anywhere un-checked detected peaks

③ Retention time window drag holding right click warping on retention time range

④ Intensity window drag holding right click warping on intensity range

Edit mode

① Left click and drag on the peak edge [red square] change the location of the peak edge

② Right click and drag detect new peak

27

Library editor [optional]

You can update the retention time and amplitude ratio of each metabolite by means of the QC

sample file or the standard mixture file You can also change the target transition for peak

quantification

The default identification is based on the highest peak in the EIC chromatogram If you need to

change the true peak double click the reverse triangle

If you change the target transition select the target MRM transition from the ldquoTarget MRMrdquo

combo box

The right-column information including the compound name target MRM RT and amplitude

ratios are generated by double-clicking the compound name in the list box on the left or by

re-selecting the true peak in the EIC chromatogram viewer

If the object metabolite is not detected click the ldquoViewrdquo button at the top left and switch to

ldquoEditrdquo mode In the ldquoEditrdquo mode hold the right mouse click and drag the peak area you want to

detect

If you want to change the retention time or amplitude information manually just type what you

want in the textbox and click the ldquoUpdaterdquo button on the top right

If you want to save the updated library click the ldquoSaverdquo button on the top left side

Lastly after editing the reference library click the ldquoFinishrdquo button on the top left side

Note The details and the operation method for chromatogram viewer are described later

28

Tool button

File start new project open existing project save as a project and save the project

Data processing for data re-processing per file per metabolite or in all data sets

Statistical analysis data normalization and statistical analysis

Window tile setting of chromatogram viewer

View sorting of chromatogram viewer

Option re-define class ID and analytical order choose the internal standard decide ldquoincluderdquo or

ldquoexcluderdquo data for statistical analysis

Identification non-meaningful in MRMPROBS project

Export The result is exported in tab-delimited text format

Help show version information

29

Tab

Chromatogram All data manipulation tasks are performed here

Raw data matrix The peak quantification value of each file and each metabolite is shown here

Processed data matrix The normalized value of each file and each metabolite is shown here

Statistical result The result of statistical analysis is shown here

Raw data matrix

30

Button

Reset Reset the display range of chromatograms

View If you push this ldquoViewrdquo button the chromatogram viewer is changed to ldquoEditrdquo mode In

the ldquoEditrdquo mode you can modify the peak edge and detect new peaks manually

None The properties of detected peaks are shown in this ComboBox You can confirm the total

score (probability) rt similarity area value and reference RT in the chromatogram viewer

Height You can set the quantification mode The default is set by peak height Instead you can

change it to area mode By using the ldquoAllrdquo option the quantification mode is reflected =

implemented in all files and all metabolites

Processed You can see raw chromatograms as well as smoothed chromatograms

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

26

② Detected window left double-click the reverse triangle change the true peak right

double-click anywhere un-checked detected peaks

③ Retention time window drag holding right click warping on retention time range

④ Intensity window drag holding right click warping on intensity range

Edit mode

① Left click and drag on the peak edge [red square] change the location of the peak edge

② Right click and drag detect new peak

27

Library editor [optional]

You can update the retention time and amplitude ratio of each metabolite by means of the QC

sample file or the standard mixture file You can also change the target transition for peak

quantification

The default identification is based on the highest peak in the EIC chromatogram If you need to

change the true peak double click the reverse triangle

If you change the target transition select the target MRM transition from the ldquoTarget MRMrdquo

combo box

The right-column information including the compound name target MRM RT and amplitude

ratios are generated by double-clicking the compound name in the list box on the left or by

re-selecting the true peak in the EIC chromatogram viewer

If the object metabolite is not detected click the ldquoViewrdquo button at the top left and switch to

ldquoEditrdquo mode In the ldquoEditrdquo mode hold the right mouse click and drag the peak area you want to

detect

If you want to change the retention time or amplitude information manually just type what you

want in the textbox and click the ldquoUpdaterdquo button on the top right

If you want to save the updated library click the ldquoSaverdquo button on the top left side

Lastly after editing the reference library click the ldquoFinishrdquo button on the top left side

Note The details and the operation method for chromatogram viewer are described later

28

Tool button

File start new project open existing project save as a project and save the project

Data processing for data re-processing per file per metabolite or in all data sets

Statistical analysis data normalization and statistical analysis

Window tile setting of chromatogram viewer

View sorting of chromatogram viewer

Option re-define class ID and analytical order choose the internal standard decide ldquoincluderdquo or

ldquoexcluderdquo data for statistical analysis

Identification non-meaningful in MRMPROBS project

Export The result is exported in tab-delimited text format

Help show version information

29

Tab

Chromatogram All data manipulation tasks are performed here

Raw data matrix The peak quantification value of each file and each metabolite is shown here

Processed data matrix The normalized value of each file and each metabolite is shown here

Statistical result The result of statistical analysis is shown here

Raw data matrix

30

Button

Reset Reset the display range of chromatograms

View If you push this ldquoViewrdquo button the chromatogram viewer is changed to ldquoEditrdquo mode In

the ldquoEditrdquo mode you can modify the peak edge and detect new peaks manually

None The properties of detected peaks are shown in this ComboBox You can confirm the total

score (probability) rt similarity area value and reference RT in the chromatogram viewer

Height You can set the quantification mode The default is set by peak height Instead you can

change it to area mode By using the ldquoAllrdquo option the quantification mode is reflected =

implemented in all files and all metabolites

Processed You can see raw chromatograms as well as smoothed chromatograms

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

27

Library editor [optional]

You can update the retention time and amplitude ratio of each metabolite by means of the QC

sample file or the standard mixture file You can also change the target transition for peak

quantification

The default identification is based on the highest peak in the EIC chromatogram If you need to

change the true peak double click the reverse triangle

If you change the target transition select the target MRM transition from the ldquoTarget MRMrdquo

combo box

The right-column information including the compound name target MRM RT and amplitude

ratios are generated by double-clicking the compound name in the list box on the left or by

re-selecting the true peak in the EIC chromatogram viewer

If the object metabolite is not detected click the ldquoViewrdquo button at the top left and switch to

ldquoEditrdquo mode In the ldquoEditrdquo mode hold the right mouse click and drag the peak area you want to

detect

If you want to change the retention time or amplitude information manually just type what you

want in the textbox and click the ldquoUpdaterdquo button on the top right

If you want to save the updated library click the ldquoSaverdquo button on the top left side

Lastly after editing the reference library click the ldquoFinishrdquo button on the top left side

Note The details and the operation method for chromatogram viewer are described later

28

Tool button

File start new project open existing project save as a project and save the project

Data processing for data re-processing per file per metabolite or in all data sets

Statistical analysis data normalization and statistical analysis

Window tile setting of chromatogram viewer

View sorting of chromatogram viewer

Option re-define class ID and analytical order choose the internal standard decide ldquoincluderdquo or

ldquoexcluderdquo data for statistical analysis

Identification non-meaningful in MRMPROBS project

Export The result is exported in tab-delimited text format

Help show version information

29

Tab

Chromatogram All data manipulation tasks are performed here

Raw data matrix The peak quantification value of each file and each metabolite is shown here

Processed data matrix The normalized value of each file and each metabolite is shown here

Statistical result The result of statistical analysis is shown here

Raw data matrix

30

Button

Reset Reset the display range of chromatograms

View If you push this ldquoViewrdquo button the chromatogram viewer is changed to ldquoEditrdquo mode In

the ldquoEditrdquo mode you can modify the peak edge and detect new peaks manually

None The properties of detected peaks are shown in this ComboBox You can confirm the total

score (probability) rt similarity area value and reference RT in the chromatogram viewer

Height You can set the quantification mode The default is set by peak height Instead you can

change it to area mode By using the ldquoAllrdquo option the quantification mode is reflected =

implemented in all files and all metabolites

Processed You can see raw chromatograms as well as smoothed chromatograms

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

28

Tool button

File start new project open existing project save as a project and save the project

Data processing for data re-processing per file per metabolite or in all data sets

Statistical analysis data normalization and statistical analysis

Window tile setting of chromatogram viewer

View sorting of chromatogram viewer

Option re-define class ID and analytical order choose the internal standard decide ldquoincluderdquo or

ldquoexcluderdquo data for statistical analysis

Identification non-meaningful in MRMPROBS project

Export The result is exported in tab-delimited text format

Help show version information

29

Tab

Chromatogram All data manipulation tasks are performed here

Raw data matrix The peak quantification value of each file and each metabolite is shown here

Processed data matrix The normalized value of each file and each metabolite is shown here

Statistical result The result of statistical analysis is shown here

Raw data matrix

30

Button

Reset Reset the display range of chromatograms

View If you push this ldquoViewrdquo button the chromatogram viewer is changed to ldquoEditrdquo mode In

the ldquoEditrdquo mode you can modify the peak edge and detect new peaks manually

None The properties of detected peaks are shown in this ComboBox You can confirm the total

score (probability) rt similarity area value and reference RT in the chromatogram viewer

Height You can set the quantification mode The default is set by peak height Instead you can

change it to area mode By using the ldquoAllrdquo option the quantification mode is reflected =

implemented in all files and all metabolites

Processed You can see raw chromatograms as well as smoothed chromatograms

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

29

Tab

Chromatogram All data manipulation tasks are performed here

Raw data matrix The peak quantification value of each file and each metabolite is shown here

Processed data matrix The normalized value of each file and each metabolite is shown here

Statistical result The result of statistical analysis is shown here

Raw data matrix

30

Button

Reset Reset the display range of chromatograms

View If you push this ldquoViewrdquo button the chromatogram viewer is changed to ldquoEditrdquo mode In

the ldquoEditrdquo mode you can modify the peak edge and detect new peaks manually

None The properties of detected peaks are shown in this ComboBox You can confirm the total

score (probability) rt similarity area value and reference RT in the chromatogram viewer

Height You can set the quantification mode The default is set by peak height Instead you can

change it to area mode By using the ldquoAllrdquo option the quantification mode is reflected =

implemented in all files and all metabolites

Processed You can see raw chromatograms as well as smoothed chromatograms

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

30

Button

Reset Reset the display range of chromatograms

View If you push this ldquoViewrdquo button the chromatogram viewer is changed to ldquoEditrdquo mode In

the ldquoEditrdquo mode you can modify the peak edge and detect new peaks manually

None The properties of detected peaks are shown in this ComboBox You can confirm the total

score (probability) rt similarity area value and reference RT in the chromatogram viewer

Height You can set the quantification mode The default is set by peak height Instead you can

change it to area mode By using the ldquoAllrdquo option the quantification mode is reflected =

implemented in all files and all metabolites

Processed You can see raw chromatograms as well as smoothed chromatograms

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

31

List Box

If you double-click a metabolite name or a file name the chromatograms are generated in the

chromatogram viewer

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

32

Details on the MRMPROBS function

File menu

New project used for creating a new project

Open project used for opening an existing project Make sure that mth file raw folder and

processed folder are included in the same directory

Save as use to save as a new file

Save use to overwrite an existing project

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

33

Data reprocessing

Data re-processing can be done by newly optimized parameters in this option Re-processing is also

performed per metabolite or per file The target MRM can also be changed The parameters are set

per metabolite and per file The required time for data re-processing is very short because file import

has been performed already

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

34

Statistical analysis

The current program can apply two types of missing value approaches and can normalize a

quantification value by the internal standard and loesscubic spline with the analytical order

information If you want to use the internal standard you must set the optimal setting in the ldquoOption

menurdquo The current program can also do principal component analysis

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

35

Missing value methods

Zero value This option gives zero (0) for ldquoNDrdquo in the raw data matrix

Data point value of retention time average This option gives the value described below for

ldquoNDrdquo in the raw data matrix

1 The process is performed per column ie per metabolite

2 If the value of a metabolite is ldquoNDrdquo in all files a zero (0) value is assigned

3 The retention time values except for ldquoNDrdquo files are stored and the average value is calculated

4 For each ldquoNDrdquo the intensity of ldquodata pointrdquo consistency with the average retention time of the

processed EIC chromatogram (after smoothing) is assigned as the quantification value

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

36

Normalization

None after implementation of the missing value approach the values of the raw data matrix are

stored in the processed data matrix

Internal standard after implementation of the missing value approach the value divided by the

internal standard value set in the ldquoOption menurdquo is stored in the processed data matrix

LOESS after implementation of the missing value approach the signal intensities of each

metabolite are normalized with the QC samples information by means of loesscubic spline

Internal standard + LOESS After internal standard normalization loesscubic spline based

normalization is performed

After clicking the ldquoDonerdquo button the ldquoStatistical analysis settingrdquo button is activated

You can do principal component analysis Add the calculated number of the principal components

and choose the scale and transform method

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

37

Zooming in and out can be done with the mouse wheel Each principal component is shown by

selecting the X axis or the Y axis combobox

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

38

Window menu

The tile setting is possible depending on your computers resolution Please select your preference

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

39

View menu

In this menu the chromatograms in the chromatogram viewer are sorted by file id analytical order

class id and file type

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

40

Option menu

Here it is possible to set the properties of metabolites and files In particular this option menu is used

to create a data matrix for statistical analysis

In the file properties you can re-set the file type class ID and analytical order except for the

file name If you clear the check box of the included property it is no longer included in the

processed data matrix

In the metabolite properties you can set the internal standard It can be set independently for

each metabolite However please make sure that the metabolite name of the internal standard is

completely consistent with the metabolite name in the ldquointernal standardrdquo column Therefore we

recommend that you use copy and paste for the internal standard setting In this window although

copy and paste can be performed just by using the keyboard you can do ldquomultirdquo copy For example

copy a metabolite name by pushing Ctrl + C Select the rows you want to add in the internal standard

column by dragging and paste the clipboard contents by pushing Ctrl + V

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

41

Export menu

A tab-delimited text file can be exported for a raw data matrix a processed data matrix the updated

library detected peak information detail and PCA results Moreover the PCA result can be exported

by some image formats

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

42

Appendix A-1 how to obtain appropriate file conversion of the Shimadzu lcd file

Although you can do a content change of the lcd file after LC-QqQMS (MRM) analysis it is very

useful to construct a suitable method file (lcm format file) for the successful file convert of the

MRMPROBS software

1 Event name and channel (MRM transitions) rule

2 Update compound table

After the method construction of MRM transitions you should update the compound table mz by

the MRM event If you can analyze the samples by using the updated method file you do not have to

perform any other tasks for the stable file convert

For stable convert of Reifycs file convert

software the compound name should be

made just by ASCII format

MRM transitions should be

constructed for one metabolite

The completely same precursor and

product mz pair cannot be

acceptable in the file converter

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

43

You can check the updated table by Method-gtData Processing Parameters-gtCompound tab

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

44

3 If your data (lcd) were not collected by a suitable method described above you can improve

the lcd file by using the method file modified in the above way After the construction of the

modified method file please open ldquoPostrun Analysisrdquo of LabSolutions

After selecting the analysis files (lcd) push the ldquoApply to Methodrdquo button

Select the modified method file and improve your lcd file including the compound table mz If you

can do this the file (lcd) is successfully converted by Reifycs Inc software

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

45

4File convert

Conditions You can convert from lcd files to abf files on your computer by installing LabSolutions

software ldquoTTFLDataExportVer5dllrdquo of LabSolutions ver 553 SP4 or later is required for the file

convert Check the ldquoTTFLDataExportVer5dllrdquo (Program Files (or 86)gtLabSolutions) file property

If the file size is less than 577536 bytes contact Shimadzu Inc for a file change

After ldquoAnalysisBaseFileConverterexerdquo is opened drag and drop the lcd files to this converter

Push the ldquoConvertrdquo button The ABF format files will be generated in the same folder as the lcd

files

Reifycs Inc software refers to this compound table for

the file convert from lcd file to abf file

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

46

Appendix B Third option of MRMPROBS via mzML file

Required software and file

MSConvert

Download link httpproteowizardsourceforgenet

MRMPROBS

Download link httpprimepscrikenjpMetabolomics_SoftwareMRMPROBSindexhtml

Reference library for compound identification (tab-delimited text file)

MRMPROBS can import the mzML format file In the third option of MRMPROBS the

ldquofunction idrdquo is utilized to extract the chromatogram data The users should add the ldquofunction idrdquo

information to the reference library in addition to the normal library format

Download ProteoWizard

1 Select download type Windows installer (includes vendor reader support) is recommended

2 Read license agreements and download the proteowizard

(httpproteowizardsourceforgenetdownloadsshtml)

Setup ProteoWizard

1 Follow the wizard windows (Maybe you donrsquot miss it)

2 ldquoSeeMSrdquo should be also imported

47

Convert the vendorrsquos MS file to mzML via ProteoWizard

1 Open the MSConvertGUIexe

2 Select ldquoList of Filesrdquo

3 Select the vendorrsquos file via ldquoBrowserdquo button

4 In the ldquoOptionsrdquo never check any additional compression including ldquoUse numpress linear

compressionrdquo ldquoUse numpress short logged float compressionrdquo and ldquoUse numpress short

positive integer compressionrdquo Each of binary encoding precision is available

5 Click ldquoStartrdquo button

Note ProteoWizard doesrsquont support Shimadzu MS format If you want to use them please use the

abf converter