FYI I am running TPP V4.4 on Windows. On Oct 22, 4:23 pm, Mike <[email protected]> wrote: > I am having a problem with using ASAP ratio to quantify mTRAQ labeled > samples. Starting with .RAW files from Thermo, I convert to mzXML, > and then convert to .mgf for database searching. I search the samples > using MASCOT with variable modifications on the n-Terminus and lysine > residues for both light and heavy tags. I convert the resulting .dat > files to pep.xml and then use PeptideProphet with XPRESS and ASAP > ratio for quantification. I set the parameters for XPRESS by changing > the labeled residues to n and K and giving the mass difference between > the light and heavy tagged residues (8 Da). I set the parameters for > ASAP by changing the labeled residues to n and K and setting the > specified residue mass of n and K to the respective monoisotopic > masses of the heavy labeled residues. In the results, XPRESS works > well. Spectra have the correct m/z and the mass difference between > the light and heavy labeled signal is correct. For example, XPRESS > associates a light labeled signal at m/z 763 with a heavy labeled > signal at 767 for a peptide sequence tagged with the light mTRAQ > reagent on the n-terminus with a mass ~1525 (m/z 763, z = 2, m =1525, > Heavy - light = 4 m/z) The results of the ASAP ratio are clearly not > correct. ASAP ratio returns the light labeled signal at 693 m/z and > the heavy labeled signal at 763 m/z for the same peptide. It appears > to have switched the light and heavy label and to use a mass > difference of heavy vs. light of 70 m/z. ASAP ratio of other peptides > are consistently nonsensical. > > It appears that I am entering the wrong parameters for the ASAP > ratio. I have tried a number of options with no success. If only the > mass of the heavy labeled residue is entered, how does the ASAP ratio > program know the mass of the light labeled residue? Similarly, if > only the difference between light and heavy residues are entered for > XPRESS, how does this program know the mass of the light and heavy > tagged residues?
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