Extracts and normalizes isobaric labeling information from an LC-MS/MS experiment.

pot. predecessor tools	→ IsobaricAnalyzer →	pot. successor tools
PeakPickerHiRes		IDMapper
FileFilter		IDMapper

The input MSn spectra have to be in centroid mode for the tool to work properly. Use e.g. PeakPickerHiRes to perform centroiding of profile data, if necessary.

This tool currently supports iTRAQ 4-plex and 8-plex, and TMT 6-plex, 10-plex, 11-plex, 16-plex, and 18-plex as labeling methods. It extracts the isobaric reporter ion intensities from centroided MS2 or MS3 data (MSn), then performs isotope correction and stores the resulting quantitation in a consensus map, in which each consensus feature represents one relevant MSn scan (e.g. HCD; see parameters select_activation and min_precursor_intensity). The MS level for quantification is chosen automatically, i.e. if MS3 is present, MS2 will be ignored. For intensity, the closest non-zero m/z signal to the theoretical position is taken as reporter ion abundance. The position (RT, m/z) of the consensus centroid is the precursor position in MS1 (from the MS2 spectrum); the consensus sub-elements correspond to the theoretical channel m/z (with m/z values of 113-121 Th for iTRAQ and 126-131 Th for TMT, respectively).

For all labeling techniques, the search radius (reporter_mass_shift) should be set as small as possible, to avoid picking up false-positive ions as reporters. Usually, Orbitraps deliver precision of about 0.0001 Th at this low mass range. Low intensity reporters might have a slightly higher deviation. By default, the mass range is set to ~0.002 Th, which should be sufficient for all instruments (~15 ppm). The tool will throw an Exception if you set it below 0.0001 Th (~0.7ppm). The tool will also throw an Exception if you set reporter_mass_shift > 0.003 Th for TMT-10plex and TMT-11plex, since this could lead to ambiguities with neighbouring channels (which are ~0.006 Th apart in most cases).

For quality control purposes, the tool reports the median distance between the theoretical vs. observed reporter ion peaks in each channel. The search radius is fixed to 0.5 Th (regardless of the user defined search radius). This allows to track calibration issues. For TMT-10plex, these results are automatically omitted if they could be confused with a neighbouring channel, i.e. exceed the tolerance to a neighbouring channel with the same nominal mass (C/N channels). If the distance is too large, you might have a m/z calibration problem (see InternalCalibration).

Note: If none of the reporter ions can be detected in an MSn scan, a consensus feature will still be generated, but the intensities of the overall feature and of all its sub-elements will be zero. (If desired, such features can be removed by applying an intensity filter in FileFilter.) However, if the spectrum is completely empty (no ions whatsoever), no consensus feature will be generated.

Isotope correction is done using non-negative least squares (NNLS), i.e.:
Minimize ||Ax - b||, subject to x >= 0, where b is the vector of observed reporter intensities (with "contaminating" isotope species), A is a correction matrix (as supplied by the manufacturer of the labeling kit) and x is the desired vector of corrected (real) reporter intensities. Other software tools solve this problem by using an inverse matrix multiplication, but this can yield entries in x which are negative. In a real sample, this solution cannot possibly be true, so usually negative values (= negative reporter intensities) are set to zero. However, a negative result usually means that noise was not properly accounted for in the calculation. We thus use NNLS to get a non-negative solution, without the need to truncate negative values. In the (usual) case that inverse matrix multiplication yields only positive values, our NNLS will give the exact same optimal solution.

The correction matrices can be found (and changed) in the INI file (parameter correction_matrix of the corresponding labeling method). However, these matrices for both 4-plex and 8-plex iTRAQ are now stable, and every kit delivered should have the same isotope correction values. Thus, there should be no need to change them, but feel free to compare the values in the INI file with your kit's certificate. For TMT (6-plex and 10-plex) the values have to be adapted for each kit: Modify the correction matrix according to the data in the product data sheet of your charge:

Data sheet:
Mass Tag  Repoter Ion -2      -1      Monoisotopic    +1     +2
126       126.12776   0.0%    0.0%        100%        5.0%   0.0%
127N      127.124761  0.0%    0.2%        100%        4.6%   0.0%
...

Corresponding correction matrix:

[0.0/0.0/5.0/0.0,
0.0/0.2/4.6/0.0,
...

After the quantitation, you may want to annotate the consensus features with corresponding peptide identifications, obtained from an identification pipeline. Use IDMapper to perform the annotation, but make sure to set suitably small RT and m/z tolerances for the mapping. Since the positions of the consensus features reported here are taken from the precursor of the MS2 (also if quant was done in MS3), it should be possible to achieve a perfect one-to-one matching of every identification (from MS2) to a single consensus feature.

Note that quantification will be solely on peptide level after this stage. In order to obtain protein quantities, you can use TextExporter to obtain a simple text format which you can feed to other software tools (e.g., R), or you can apply ProteinQuantifier.

The command line parameters of this tool are:

IsobaricAnalyzer -- Calculates isobaric quantitative values for peptides
Full documentation: http://www.openms.de/doxygen/release/3.2.0/html/TOPP_IsobaricAnalyzer.html
Version: 3.2.0 Nov 18 2024, 16:14:00, Revision: 03223c3
To cite OpenMS:
 + Pfeuffer, J., Bielow, C., Wein, S. et al.. OpenMS 3 enables reproducible analysis of large-scale mass spec
   trometry data. Nat Methods (2024). doi:10.1038/s41592-024-02197-7.

Usage:
  IsobaricAnalyzer <options>

This tool has algorithm parameters that are not shown here! Please check the ini file for a detailed descript
ion or use the --helphelp option

Options (mandatory options marked with '*'):
  -type <mode>       Isobaric Quantitation method used in the experiment. (default: 'itraq4plex') (valid: 
                     'itraq4plex', 'itraq8plex', 'tmt10plex', 'tmt11plex', 'tmt16plex', 'tmt18plex', 'tmt6ple
                     x')
  -in <file>*        Input raw/picked data file  (valid formats: 'mzML')
  -out <file>*       Output consensusXML file with quantitative information (valid formats: 'consensusXML')
                     
Common TOPP options:
  -ini <file>        Use the given TOPP INI file
  -threads <n>       Sets the number of threads allowed to be used by the TOPP tool (default: '1')
  -write_ini <file>  Writes the default configuration file
  --help             Shows options
  --helphelp         Shows all options (including advanced)

The following configuration subsections are valid:
 - extraction       Parameters for the channel extraction.
 - itraq4plex       Algorithm parameters for iTRAQ 4-plex
 - itraq8plex       Algorithm parameters for iTRAQ 8-plex
 - quantification   Parameters for the peptide quantification.
 - tmt10plex        Algorithm parameters for TMT 10-plex
 - tmt11plex        Algorithm parameters for TMT 11-plex
 - tmt16plex        Algorithm parameters for TMT 16-plex
 - tmt18plex        Algorithm parameters for TMT 18-plex
 - tmt6plex         Algorithm parameters for TMT 6-plex

You can write an example INI file using the '-write_ini' option.
Documentation of subsection parameters can be found in the doxygen documentation or the INIFileEditor.
For more information, please consult the online documentation for this tool:
  - http://www.openms.de/doxygen/release/3.2.0/html/TOPP_IsobaricAnalyzer.html

INI file documentation of this tool:

Legend:

required parameter

advanced parameter

+IsobaricAnalyzerCalculates isobaric quantitative values for peptides

version3.2.0 Version of the tool that generated this parameters file.

++1Instance '1' section for 'IsobaricAnalyzer'

typeitraq4plex Isobaric Quantitation method used in the experiment.itraq4plex, itraq8plex, tmt10plex, tmt11plex, tmt16plex, tmt18plex, tmt6plex

in input raw/picked data file input file*.mzML

out output consensusXML file with quantitative informationoutput file*.consensusXML

log Name of log file (created only when specified)

debug0 Sets the debug level

threads1 Sets the number of threads allowed to be used by the TOPP tool

no_progressfalse Disables progress logging to command linetrue, false

forcefalse Overrides tool-specific checkstrue, false

testfalse Enables the test mode (needed for internal use only)true, false

+++extractionParameters for the channel extraction.

select_activationauto Operate only on MSn scans where any of its precursors features a certain activation method. Setting to "auto" uses HCD and HCID spectra. Set to empty string if you want to disable filtering.auto, Collision-induced dissociation, Post-source decay, Plasma desorption, Surface-induced dissociation, Blackbody infrared radiative dissociation, Electron capture dissociation, Infrared multiphoton dissociation, Sustained off-resonance irradiation, High-energy collision-induced dissociation, Low-energy collision-induced dissociation, Photodissociation, Electron transfer dissociation, Electron transfer and collision-induced dissociation, Electron transfer and higher-energy collision dissociation, Pulsed q dissociation, trap-type collision-induced dissociation, beam-type collision-induced dissociation, in-source collision-induced dissociation, any

reporter_mass_shift2.0e-03 Allowed shift (left to right) in Th from the expected position.1.0e-04:0.5

min_precursor_intensity1.0 Minimum intensity of the precursor to be extracted. MS/MS scans having a precursor with a lower intensity will not be considered for quantitation.0.0:∞

keep_unannotated_precursortrue Flag if precursor with missing intensity value or missing precursor spectrum should be included or not.true, false

min_reporter_intensity0.0 Minimum intensity of the individual reporter ions to be extracted.0.0:∞

discard_low_intensity_quantificationsfalse Remove all reporter intensities if a single reporter is below the threshold given in 'min_reporter_intensity'.true, false

min_precursor_purity0.0 Minimum fraction of the total intensity in the isolation window of the precursor spectrum attributable to the selected precursor.0.0:1.0

precursor_isotope_deviation10.0 Maximum allowed deviation (in ppm) between theoretical and observed isotopic peaks of the precursor peak in the isolation window to be counted as part of the precursor.0.0:∞

purity_interpolationtrue If set to true the algorithm will try to compute the purity as a time weighted linear combination of the precursor scan and the following scan. If set to false, only the precursor scan will be used.true, false

+++itraq4plexAlgorithm parameters for iTRAQ 4-plex

channel_114_description Description for the content of the 114 channel.

channel_115_description Description for the content of the 115 channel.

channel_116_description Description for the content of the 116 channel.

channel_117_description Description for the content of the 117 channel.

reference_channel114 Number of the reference channel (114-117).114:117

correction_matrix[0.0/1.0/5.9/0.2, 0.0/2.0/5.6/0.1, 0.0/3.0/4.5/0.1, 0.1/4.0/3.5/0.1] Correction matrix for isotope distributions (see documentation); use the following format: <-2Da>/<-1Da>/<+1Da>/<+2Da>; e.g. '0/0.3/4/0', '0.1/0.3/3/0.2'

+++itraq8plexAlgorithm parameters for iTRAQ 8-plex

channel_113_description Description for the content of the 113 channel.

channel_114_description Description for the content of the 114 channel.

channel_115_description Description for the content of the 115 channel.

channel_116_description Description for the content of the 116 channel.

channel_117_description Description for the content of the 117 channel.

channel_118_description Description for the content of the 118 channel.

channel_119_description Description for the content of the 119 channel.

channel_121_description Description for the content of the 121 channel.

reference_channel113 Number of the reference channel (113-121). Please note that 120 is not valid.113:121

correction_matrix[0.00/0.00/6.89/0.22, 0.00/0.94/5.90/0.16, 0.00/1.88/4.90/0.10, 0.00/2.82/3.90/0.07, 0.06/3.77/2.99/0.00, 0.09/4.71/1.88/0.00, 0.14/5.66/0.87/0.00, 0.27/7.44/0.18/0.00] Correction matrix for isotope distributions (see documentation); use the following format: <-2Da>/<-1Da>/<+1Da>/<+2Da>; e.g. '0/0.3/4/0', '0.1/0.3/3/0.2'

+++quantificationParameters for the peptide quantification.

isotope_correctiontrue Enable isotope correction (highly recommended). Note that you need to provide a correct isotope correction matrix otherwise the tool will fail or produce invalid results.true, false

normalizationfalse Enable normalization of channel intensities with respect to the reference channel. The normalization is done by using the Median of Ratios (every channel / Reference). Also the ratio of medians (from any channel and reference) is provided as control measure!true, false

+++tmt10plexAlgorithm parameters for TMT 10-plex

channel_126_description Description for the content of the 126 channel.

channel_127N_description Description for the content of the 127N channel.

channel_127C_description Description for the content of the 127C channel.

channel_128N_description Description for the content of the 128N channel.

channel_128C_description Description for the content of the 128C channel.

channel_129N_description Description for the content of the 129N channel.

channel_129C_description Description for the content of the 129C channel.

channel_130N_description Description for the content of the 130N channel.

channel_130C_description Description for the content of the 130C channel.

channel_131_description Description for the content of the 131 channel.

reference_channel126 The reference channel (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131).126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131

correction_matrix[0.0/0.0/5.09/0.0, 0.0/0.25/5.27/0.0, 0.0/0.37/5.36/0.15, 0.0/0.65/4.17/0.1, 0.08/0.49/3.06/0.0, 0.01/0.71/3.07/0.0, 0.0/1.32/2.62/0.0, 0.02/1.28/2.75/2.53, 0.03/2.08/2.23/0.0, 0.08/1.99/1.65/0.0] Correction matrix for isotope distributions (see documentation); use the following format: <-2Da>/<-1Da>/<+1Da>/<+2Da>; e.g. '0/0.3/4/0', '0.1/0.3/3/0.2'

+++tmt11plexAlgorithm parameters for TMT 11-plex