Hi there, I am trying to do run a quantitative analysis of dimethylated samples labelled on N-term and lysine (using either trypsin or ArgC) using TPP ver5 on Windows. I ran X!Tandem or Comet searches using fixed light di-Met modification of Lys and N-term (+28.03) and the Heavy-Light delta (6.031) as variable modifications as explained here, and I used successfully before. Quantification with XPRESS run without any error but the ratio calculation seems to be correct only for peptides that contain a lysine (regardless if they were heavy or light). The ratio calculated for peptides that do not contain lysines is correct only for the light labelled ones but for the heavy ones the program seems to consider them as light peptides and searches for peaks that are +6 heavier in order to calculate the ratio. For example if we got the peptide AAAHTSPR that was identified in the light dimethylation on the N-term, MH= 838.4530) the program will look for the heavy couple at the right place (MH=844.4848) but if the same peptide was identified in its heavy form (MH 838.4530) it seems like the program is looking for the heavy partner at 844.4848. Another minor (?) comment is that running such searches with X!tandem seems to mark the heavy N-term dimethylation in a different way than it used to before (and currently appear in Comet pepxml) add looks like this:n7.04A 99.07TAGDTHLGGEDFDNR (instead of n35.07 <http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?CMD=Web&LAYOUT=TwoWindows&AUTO_FORMAT=Semiauto&ALIGNMENTS=50&ALIGNMENT_VIEW=Pairwise&CDD_SEARCH=on&CLIENT=web&COMPOSITION_BASED_STATISTICS=on&DATABASE=nr&DESCRIPTIONS=100&ENTREZ_QUERY=%28none%29&EXPECT=1000&FILTER=L&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&I_THRESH=0.005&MATRIX_NAME=BLOSUM62&NCBI_GI=on&PAGE=Proteins&PROGRAM=blastp&SERVICE=plain&SET_DEFAULTS.x=41&SET_DEFAULTS.y=5&SHOW_OVERVIEW=on&END_OF_HTTPGET=Yes&SHOW_LINKOUT=yes&QUERY=ATAGDTHLGGEDFDNR> ATAGDTHLGGEDFDNR. <http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?CMD=Web&LAYOUT=TwoWindows&AUTO_FORMAT=Semiauto&ALIGNMENTS=50&ALIGNMENT_VIEW=Pairwise&CDD_SEARCH=on&CLIENT=web&COMPOSITION_BASED_STATISTICS=on&DATABASE=nr&DESCRIPTIONS=100&ENTREZ_QUERY=%28none%29&EXPECT=1000&FILTER=L&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&I_THRESH=0.005&MATRIX_NAME=BLOSUM62&NCBI_GI=on&PAGE=Proteins&PROGRAM=blastp&SERVICE=plain&SET_DEFAULTS.x=41&SET_DEFAULTS.y=5&SHOW_OVERVIEW=on&END_OF_HTTPGET=Yes&SHOW_LINKOUT=yes&QUERY=ATAGDTHLGGEDFDNR> I hope you can advise. Thanks Oded
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