Extracts and normalizes isobaric labeling information from an LC-MS/MS experiment.
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 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).
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.
Legend:
required parameter
advanced parameter
+IsobaricAnalyzerCalculates isobaric quantitative values for peptides
version3.4.0-pre-nightly-2024-12-16
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
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_131N_description
Description for the content of the 131N channel.
channel_131C_description
Description for the content of the 131C channel.
reference_channel126
The reference channel (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C).126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C
correction_matrix[0.0/0.0/8.6/0.3, 0.0/0.1/7.8/0.1, 0.0/0.8/6.9/0.1, 0.0/7.4/7.4/0.0, 0.0/1.5/6.2/0.2, 0.0/1.5/5.7/0.1, 0.0/2.6/4.8/0.0, 0.0/2.2/4.6/0.0, 0.0/2.8/4.5/0.1, 0.1/2.9/3.8/0.0, 0.0/3.9/2.8/0.0]
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'
+++tmt16plexAlgorithm parameters for TMT 16-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_131N_description
Description for the content of the 131N channel.
channel_131C_description
Description for the content of the 131C channel.
channel_132N_description
Description for the content of the 132N channel.
channel_132C_description
Description for the content of the 132C channel.
channel_133N_description
Description for the content of the 133N channel.
channel_133C_description
Description for the content of the 133C channel.
channel_134N_description
Description for the content of the 134N channel.
reference_channel126
The reference channel (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C, 132N, 132C, 133N, 133C, 134N).126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C, 132N, 132C, 133N, 133C, 134N
correction_matrix[NA/NA / NA/NA / 0.31/9.09 / 0.02/0.32, NA/NA / NA/0.78 / NA/9.41 / NA/0.33, NA/NA / 0.93/NA / 0.35/8.63 / 0.01/0.27, NA/0.00 / 0.82/0.65 / NA/8.13 / NA/0.26, 0.00/NA / 1.47/NA / 0.34/6.91 / 0.00/0.15, 0.00/0.00 / 1.46/1.28 / NA/6.86 / NA/0.15, 0.13/NA / 2.59/NA / 0.32/6.07 / 0.1/0.09, 0.13/0.00 / 2.41/0.27 / NA/5.58 / NA/0.10, 0.04/NA / 3.10/NA / 0.42/4.82 / 0.02/0.06, 0.03/0.00 / 2.78/0.63 / NA/4.57 / NA/0.12, 0.08/NA / 3.90/NA / 0.47/3.57 / 0.00/0.04, 0.15/0.01 / 3.58/0.72 / NA/1.80 / NA/0.00, 0.11/NA / 4.55/NA / 0.43/1.86 / 0.00/0.00, 0.07/0.01 / 3.14/0.73 / NA/3.40 / NA/0.03, 0.22/NA / 4.96/NA / 0.34/1.03 / 0.00/NA, 0.30/0.03 / 5.49/0.62 / NA/1.14 / NA/NA]
Correction matrix for isotope distributions in percent from the Thermo data sheet (see documentation); Please provide 16 entries (rows), separated by comma, where each entry contains 8 values in the following format: <-2C13>/<-N15-C13>/<-C13>/<-N15>/<+N15>/<+C13>/<+N15+C13>/<+2C13> e.g. one row may look like this: 'NA/0.00 / 0.82/0.65 / NA/8.13 / NA/0.26'. You may use whitespaces at your leisure to ease reading.
+++tmt18plexAlgorithm parameters for TMT 18-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_131N_description
Description for the content of the 131N channel.
channel_131C_description
Description for the content of the 131C channel.
channel_132N_description
Description for the content of the 132N channel.
channel_132C_description
Description for the content of the 132C channel.
channel_133N_description
Description for the content of the 133N channel.
channel_133C_description
Description for the content of the 133C channel.
channel_134N_description
Description for the content of the 134N channel.
channel_134C_description
Description for the content of the 134C channel.
channel_135N_description
Description for the content of the 135N channel.
reference_channel126
The reference channel (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C, 132N, 132C, 133N, 133C, 134N, 134C, 135N).126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C, 132N, 132C, 133N, 133C, 134N, 134C, 135N
correction_matrix[NA/NA /NA/NA /0.31/9.09 /0.02/0.32, NA/NA /NA/0.78 /NA/9.41 /NA/0.33, NA/NA /0.93/NA /0.35/8.63 /0.01/0.27, NA/0.00 /0.82/0.65 /NA/8.13 /NA/0.26, 0.00/NA /1.47/NA /0.34/6.91 /0.00/0.15, 0.00/0.00 /1.46/1.28 /NA/6.86 /NA/0.15, 0.13/NA /2.59/NA /0.32/6.07 /0.1/0.09, 0.13/0.00 /2.41/0.27 /NA/5.58 /NA/0.10, 0.04/NA /3.10/NA /0.42/4.82 /0.02/0.06, 0.03/0.00 /2.78/0.63 /NA/4.57 /NA/0.12, 0.08/NA /3.90/NA /0.47/3.57 /0.00/0.04, 0.15/0.01 /3.58/0.72 /NA/1.80 /NA/0.00, 0.11/NA /4.55/NA /0.43/1.86 /0.00/0.00, 0.07/0.01 /3.14/0.73 /NA/3.40 /NA/0.03, 0.22/NA /4.96/NA /0.34/1.03 /0.00/NA, 0.30/0.03 /5.49/0.62 /NA/1.14 /NA/NA, 0.14/NA /5.81/NA /0.31/NA /NA/NA, 0.19/0.02 /5.42/0.36 /NA/NA /NA/NA]
Correction matrix for isotope distributions in percent from the Thermo data sheet (see documentation); Please provide 18 entries (rows), separated by comma, where each entry contains 8 values in the following format: <-2C13>/<-N15-C13>/<-C13>/<-N15>/<+N15>/<+C13>/<+N15+C13>/<+2C13> e.g. one row may look like this: 'NA/0.00 / 0.82/0.65 / NA/8.13 / NA/0.26'. You may use whitespaces at your leisure to ease reading.
+++tmt6plexAlgorithm parameters for TMT 6-plex
channel_126_description
Description for the content of the 126 channel.
channel_127_description
Description for the content of the 127 channel.
channel_128_description
Description for the content of the 128 channel.
channel_129_description
Description for the content of the 129 channel.
channel_130_description
Description for the content of the 130 channel.
channel_131_description
Description for the content of the 131 channel.
reference_channel126
Number of the reference channel (126-131).126:131
correction_matrix[0.0/0.0/8.6/0.3, 0.0/0.1/7.8/0.1, 0.0/1.5/6.2/0.2, 0.0/1.5/5.7/0.1, 0.0/3.1/3.6/0.0, 0.1/2.9/3.8/0.0]
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'