Extracts and normalizes iTRAQ information from an MS experiment.
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Extract the iTRAQ reporter ion intensities (4plex or 8plex) from raw MS2 data, does isotope corrections and stores the resulting quantitation as consensusXML, where each consensus centroid corresponds to one iTRAQ 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).
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 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.
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:
INI file documentation of this tool:
OpenMS / TOPP release 2.0.0 | Documentation generated on Sat Feb 20 2016 12:42:28 using doxygen 1.8.10 |