Extracts and normalizes isobaric labeling information from an MS experiment.
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Extract the isobaric reporter ion intensities (currently iTRAQ 4plex and 8plex and TMT 6plex are supported) from raw MS2 data, does isotope corrections and stores the resulting quantitation as consensusXML, where each consensus centroid corresponds to one isobaric MS2 scan (e.g., HCD). The position of the centroid is the precursor position, its sub-elements are the channels (thus having m/z's of 113-121 for iTRAQ 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 AB Sciex) and x is the desired vector of corrected (real) reporter intensities. Other software solves this problem 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 0. However, a negative result usually means, that noise was not accounted for thus we 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. However, these matrices for both 4plex and 8plex 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 6plex the values have to be adapted for each kit.
After this quantitation step, you might want to annotate the consensus elements with the respective identifications, obtained from an identification pipeline. Note that quantification is solely on peptide level at this stage. In order to obtain protein quantifications, you can try TextExporter to obtain a simple text format which you can feed to other software tools (e.g., R), or you can try ProteinQuantifier.
The command line parameters of this tool are:
IsobaricAnalyzer -- Calculates isobaric quantitative values for peptides Version: 1.11.1 Nov 14 2013, 11:18:15, Revision: 11976 Usage: IsobaricAnalyzer <options> This tool has algoritm parameters that are not shown here! Please check the ini file for a detailed descripti on 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', '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 /Users/aiche/dev/openms/openms-1.11.1/share/OpenMS/IDPool/IDPool.txt) --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 itraq4plex - itraq8plex Algorithm parameters for itraq8plex - quantification Parameters for the peptide quantification. - tmt6plex Algorithm parameters for tmt6plex 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 1.11.1 | Documentation generated on Thu Nov 14 2013 11:19:24 using doxygen 1.8.5 |