Getting started

Dependencies

In order to run IsobaricQuant Java 17 is needed, first of all make sure you don’t have the correct version installed already with the following command:

java --version

If you don’t have Java 17 installed follow the official instructions on how to install Java 17 for your platform:

Microsoft Windows

Installation of the JDK on Microsoft Windows Platforms: Instructions include steps to install the JDK on 64-bit Microsoft Windows operating systems.

Linux

Installation of the JDK on Linux Platforms: Instructions include installation from Archive files and Red Hat Package Manager (RPM).

macOS

Installation of the JDK on macOS: Instructions include steps to install the JDK on the macOS platform.

Installation

Since IsobaricQuant is a java application, its installation is straightforward, first you need to download the jar file for your platform:

Once the jar file is downloaded, place it in a folder of your choice, open a terminal and navigate to the jar file location. That’s it!

How to run IsobaricQuant?

After downloading the jar file, IsobaricQuant can be used via GUI:

java --add-opens java.base/java.lang=ALL-UNNAMED -jar IsobaricQuant.jar

Or it can be used via command line:

java -jar IsobaricQuant.jar -c <config_file> -mzf <mz_file> -h <hits_file> -o <output_folder>

IsobaricQuant CLI mode

java -jar IsobaricQuant.jar -c <config_file> -mzf <mz_file> -h <hits_file> -o <output_folder>

The CLI mode doesn’t include a viewer, as soon as the command is executed the quantification starts, once is completed an output folder is generated with all the output files detailed in the previous section. The generated files can be opened using the GUI version at any time. The CLI version will allow the user to include IsobaricQuant in any pipeline

IsobaricQuant GUI mode

On the GUI mode you will find a window with some options:

  1. Select the input files, for more information about input files refer to the Input/Output Files section

  2. Once all the files have been selected preceed with the quantification using the Quantify button

  3. When the quantification is completed you can proceed to consult the peptide information

_images/isobaricquant_win_01.png

In the log window you can check if the path of the selected files is correct:

_images/isobaricquant_win_02.png

Once you click the Quantify button, the quantification will start, indicating the steps involved on the process:

_images/isobaricquant_win_03.png

When the quantification is done, an output folder is created with the quantification information. The GUI includes a viewer, that will allow the user to view the quantification matching information including fragment ions

_images/isobaricquant_win_04.png _images/isobaricquant_win_05.png _images/isobaricquant_win_06.png _images/isobaricquant_win_07.png

Input Files

Configuration file

{
    "generateFragmentIonsFile": true,
    "ms2NoiseWindowTolerance": 1,
    "searchFragmentWIndowTolerance": 1.5,
    "isoMethod": "TMTpro",
    "confLabels": "all",
    "PPMTolerance": 20,
    "MS1PPMTolerance": 20,
    "MS2PPMTolerance": 20,
    "massType": "HCD_FRAGMENTATION",
    "scanLevel": 2,
    "scoreType": "ISOTOPE_DISTRIBUTION",
    "ms1PrecWindowDaltons": 0.35,
    "neutralLossesFI": "63.98,18.01,17.03",
    "neutralLosses": "63.98,18.01,17.03",
    "topXNum": 10,
    "ms1DepthSearch": 5,
    "recalcIntensities": false,
    "dataSheetName": "TMT11_TL277832",
    "modifications": {
        "varMods": "15.9949146202 M 42.01056468472 n",
        "consMods": "pep_n 304.2071453165, C 57.02146372118, K 304.2071453165",
        "varTermParams": ""
    },
    "mzFilePath": "",
    "hitImpurity": "master_scan",
    "searchMethod": "most_intense"
}

Parameter description

isoMethod: Isobaric Methods
Values: iTRAQ4plex,iTRAQ8plex,TMTDuplex,TMT6plex,TMT10plex,TMT11plex,TMTpro
Default: iTRAQ4plex
massType: mass type
Values: HCD fragmentation,Neutral mass
Default: HCD fragmentation
scanLevel: Scan level for isobaric quantification
Values: MS2,MS3
Default: MS2
scoreType: Score calculation
Values:
Isotope distribution: peptide isotopes intensity/total intensity in isotopes window.
Reporters intensity: reporters intensity/total intensity in window.
Reporters found: num. reporters found/num. Reporters
Default: Reporters intensity
confLabels: Labels to be searched in (use comma separated values. ex: 114,115,116,117). Write ‘all’ to search all of them. For TMT10plex, use label reagent: 126,127N,127C,128N,128C…
Values: input by user
Default: all
recalcIntensities: If activated, it will recalculate intensities using the product data sheet.
Default: not activated
dataSheetName: Product data sheet
Values: TMT10plex_QI218066,TMT11_TL277832
Default: TMT10plex_QI218066
PPMTolerance: PPM Tolerance for MS3
Values: input by user
Default: 10
MS1PPMTolerance: PPM Tolerance for MS1
Values: input by user
Default: 10
MS2PPMTolerance: PPM Tolerance for MS2
Values: input by user
Default: 1000
ms1PrecWindowDaltons: Dalton window that will be used to calculate the precursor signal percentage in the MS1 scan.
Values: input by user
Default: 1
searchFragmentWindowTolerance: MS2 precursor window (Da)
Values: input by user
Default: 2.5
generateFragmentIonsFile: Generates a file with all peptides’ fragment ions.
Default: not activated
hitImpurity:
Values: master_scan,weighted_avg
The precursor signal is calculated using the weighted signal or signal percentage
If it’s not weighted avg, then it will be precursor int / intensities in isolation window (calculated using ms1PrecWindowDaltons)
signalPerc = precIntensity / allIntensities

if it’s weighted, then it will calculate the same thing as the above but for the current ms1, its previous ms1 and its posterior ms1. The result is the weighted signal, calculated using the RT distance to the current ms1

(dPivotCurrent / totalDistances) * currentSignal + (dPivotPre / totalDistances) * preSignal + (dPivotPost / totalDistances) * postSignal

Default: master_scan

searchMethod: Search method used to select a TMT reporter ion peak
Values: most_intense, least_intense, lower_ppm_error
Default: most_intense
ms1DepthSearch: Number of MS1 scans depth to search for MS3 scans
Values: input by user
Default: 5
topXNum: Top X most intense ions used to calculate scores
Values: input by user
Default: 10
neutralLosses: Comma separated neutral loss masses (ex: 97.977,97.995). The peak will be searched for these NL.
Values: input by user, e.g. “63.98,18.01,17.03”
neutralLossesFI: Comma separated neutral loss masses (ex: 97.977,97.995). The fragment ions will be searched for these NL.
Values: input by user, e.g. “63.98,18.01,17.03”

Mz File

mzML or mzXML input file. The instrument raw file can be converted from existing tools such as msconvert.

Hits file

Comma separated values of the identified peptides. A conversion of the output file of a search algorithm such as Comet is required.

The values are the following:

Search ID: integer value to identify the search where the peptide was detected.
Peptide ID: integer value that identifies the peptide
Sequence: peptide sequence
Reference: protein sequence reference
Charge: peptide charge
Start Scan: peptide MS2 Scan
M/Z: peptide m/z

Output Files

Once IsobaricQuant finishes the quantification an output folder will be created on the same folder where the IsobaricQuant jar file resides.

The output folder contains 4 csv files with the result of the quantification

isofrag.csv

File containing matching information for the peptide’s fragment ions

Peptide ID: peptide id introduced in the input file
Quant ID: quantification id (unused field at 0)
Fragment ion charge
Fragment ion type (a, b…)
Fragment ion position
Fragment ion mz
Fragment ion mz difference
Fragment ion intensity
Fragment ion matched (true or false)

isolab.csv

File containing the quantification data

Peptide ID: peptide id introduced in the input file
Quant ID: quantification id (unused field at 0)
Label ID: string identificator of the label
Label MZ: Matching peak MZ (theoretical if nothing matched)
MZ Variance: mz variance between the theoretical mz and the matched mz (0 otherwise)
Scan: matched scan number
Scan: matched scan number (duplicated)
Number of scans: 1 default value
Unused parameter: 0 default value
Unused parameter: 0 default value
Label intensity: Matching peak intensity (0 otherwise)
MS2 Scan: MS2 scan introduced in the input file
Retention Time: matched scan retention time

isopep.csv

File containing the basic information to be used to match the results with the other files

Quant ID: quantification id (unused field at 0)
Peptide ID: peptide id introduced in the input file
Search ID: search id introduced in the input file
MS2 Scan: MS2 scan introduced in the input file
Noise: Calculated noise in the scan
Score: calculated score as specified in the configuration file (isotope distribution, reporters intensity or reporters found)

isopep_extra.csv

File containing the quantification scores. Please, check the scores description for further information.

Peptide ID: peptide id introduced in the input file
Quant ID: quantification id (unused field at 0)
Precursor signal
PeptideIntensityScore
MS2 TopXPeptidePeaksRatio
MS2 PeptideTopXIntensityScore
MS2 TopXIntensityFromTotalScore
MS2 PeptideTopXIntensityFromTotalScore
MS2 TopPeakIntensityScore
MS2 isTopPeakFromPeptide: 1 (yes), 0 (no)
MS2 isTopPeakFromPeptideNeutralLoss(): 1 (yes), 0 (no)
MS2 TopPeakIntensityTopXScore()
MS2 TopPeakMass
msnTotalSignal
precTPIntRatio
precRepIntRatio
precRepIntRatio
precTotalSignal
totalSignalSPSWind
Scan level: 2 or 3
MS1 scan number: 0 if not found
MS2 scan number
MS3 scan number: 0 if not found or the selected level was 2
MS1 retention time: 0 if not found
MS2 retention time
MS3 retention time: 0 if not found or the selected level was 2
SPS masses: their mz separated by ;

Test files

Once IsobaricQuant is ready to run, you can use the following input and output sample files to test the jar and its dependencies are working properly

Input files

Output files