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IsobaricAnalyzer

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

pot. predecessor tools $ \longrightarrow $ IsobaricAnalyzer $ \longrightarrow $ pot. successor tools
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This tool currently supports iTRAQ 4-plex and 8-plex, and TMT 6-plex and 10-plex as labeling methods. It extracts the isobaric reporter ion intensities from raw MS2 data, performs isotope correction and stores the resulting quantitation in a consensus map, in which each consensus feature represents one relevant MS2 scan (e.g. HCD; see parameters select_activation and min_precursor_intensity). The position (RT, m/z) of the consensus centroid is the precursor position; the sub-elements correspond to the channels (with m/z values of 113-121 for iTRAQ and 126-131 for TMT, respectively).

Note
If none of the reporter ions can be detected in an MS2 scan, a consensus feature will still be generated, but the intensities of the overall feature and of all its sub-elements will be zero.

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.

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 identifications will come from the very same MS2 scans that are now represented by consensus features. In general it should be possible to achieve a perfect one-to-one matching of every identification 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
Version: 2.0.0 May 16 2015, 09:22:21, Revision: GIT-NOTFOUND

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: 'itra
                     q4plex', 'itraq8plex', 'tmt10plex', 'tmt6plex')
  -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
  -id_pool <file>    ID pool file to DocumentID's for all generated output files. Disabled by default. (Set 
                     to 'main' to use /builddir/build/BUILD/OpenMS-Release2.0.0/share/OpenMS/IDPool/IDPool.tx
                     t)
  --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
 - 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.
Have a look at the OpenMS documentation for more information.

INI file documentation of this tool:

Legend:
required parameter
advanced parameter
+IsobaricAnalyzerCalculates isobaric quantitative values for peptides
version2.0.0 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,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 Overwrite tool specific checks.true,false
id_pool ID pool file to DocumentID's for all generated output files. Disabled by default. (Set to 'main' to use /builddir/build/BUILD/OpenMS-Release2.0.0/share/OpenMS/IDPool/IDPool.txt)
testfalse Enables the test mode (needed for internal use only)true,false
+++extractionParameters for the channel extraction.
select_activationHigh-energy collision-induced dissociation Operate only on MSn scans where any of its precursors features a certain activation method (e.g., usually HCD for iTRAQ). Set to empty string if you want to disable filtering.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,
reporter_mass_shift0.1 Allowed shift (left to right) in Da from the expected position.1e-08:0.5
min_precursor_intensity1 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:∞
keep_unannotated_precursortrue Flag if precursor with missing intensity value or missing precursor spectrum should be included or not.true,false
min_reporter_intensity0 Minimum intensity of the individual reporter ions to be extracted.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 Minimum fraction of the total intensity in the isolation window of the precursor spectrum attributable to the selected precursor.0:1
precursor_isotope_deviation10 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:∞
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[0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0] 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'
+++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[0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0, 0.0/0.0/0.0/0.0] 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'

OpenMS / TOPP release 2.0.0 Documentation generated on Sat May 16 2015 16:13:42 using doxygen 1.8.9.1