Hi- I'm attempting to use ASAPratio and Xpress to quantify SILAC samples. In my first attempts, I get very few peptides considered for quantitation by ASAPratio but hundreds by Xpress. But my Xpress st.dev. are high. I have many heavy and light versions of the peptides identified, but am unsure of why they aren't being used for quantitation. I'm trying to optimize this and in searching through many posts on this blog, it seems that some folks recommend doing two separate Sequest searches for SILAC data. Is this still the recommendation? To date, I had done only a single search allowing heavy R/K as differential modifications and did not use the 'static run" option for ASAPratio. Can someone give me advice on whether my params are correct for future processing? Also, I"m unsure of whether to include the C and N mods in ASAPratioi specified residue masses.
I'm using an Orbitrap. I have R(10) & K(8) in my heavy sample in addition to carbamidomethylation (C) and deamidation on (N) - (the result of deglycosylation). My proposed parameters: Sequest searching: search #1. Static mod on C(160.030) and differential on N(115.026) - leaving R/K to be normal search #2. Static mods on R(166.10938), K(136.10916), C(160.030), and differential on N(115.026) XPress processing: Mass tolerance: 0.01 Residue mass difference: K(8.01419) & R(10.008269) ASAPratio processing: Check static run modification - but do I use C, K, R for changed labeled residues?? Check use fixed scan range for heavy/light m/z range: 0.1 Specified residue Masses: R(166.10938), K(136.10916), C(160.030), N(115.026) - or just R/K? Any help would be much appreciated. Thanks! Rebekah -- 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.
