Extracts and normalizes isobaric labeling information from an LC-MS/MS experiment.
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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).
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 29 2015, 13:57:09, 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:
OpenMS / TOPP release 2.0.0 | Documentation generated on Fri May 29 2015 17:20:34 using doxygen 1.8.9.1 |