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? -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To post to this group, send email to [email protected]. To unsubscribe from this group, send email to [email protected]. For more options, visit this group at http://groups.google.com/group/spctools-discuss?hl=en.
