About Modification

[lbwfff]

Hi I want to analyze TMT11 mass spectrum using MS-GF+ and I have some confusion about the choice of modification. I would like to add carbamidomethylation onCys residues and TMT modification on the peptide N-terminus and Lys residues, and dynamic oxidation on Met residues. But I see that there are two options, Static (fixed) modifications and Variable Modifications, which should I put the TMT modification on? Thanks, LeeLee

[FarmGeek4Life]

Usually the TMT modifications are fixed, unless you have concerns about the TMT labelling efficiency and are trying to salvage the results.

[alchemistmatt]

This parameter file has what you want:

• MSGFPlus_PartTryp_DynMetOx_Stat_CysAlk_TMT_6Plex_20ppmParTol.txt
  • The name says "6plex" because it has the TMT6plex modification (+229.1629) on K and the N-terminus. This is because 6-plex, 10-plex, and 11-plex TMT all use +229.1629.
• If your FASTA file is large (or to greatly speed up the search), change NTT to NTT=2

For information on using parameter files, see the documentation

[lbwfff]

This parameter file has what you want:

• MSGFPlus_PartTryp_DynMetOx_Stat_CysAlk_TMT_6Plex_20ppmParTol.txt

  • The name says "6plex" because it has the TMT6plex modification (+229.1629) on K and the N-terminus. This is because 6-plex, 10-plex, and 11-plex TMT all use +229.1629.
• If your FASTA file is large (or to greatly speed up the search), change NTT to NTT=2

For information on using parameter files, see the documentation

Hi, Thank you for your reply, I also have a question that may not be software related. The "-ntt 1" parameter means that trypsin is not accurate cleavage at one termini of the protein (if I understand it correctly), I would like to ask if this is occur frequently in proteomes? Thanks, LeeLee

[alchemistmatt]

People use -ntt 1 to look for "missed" cleavages, meaning, as you guessed, that trypsin did not cleave at the given residue. In most cases, this is true for 5% or fewer peptides, but there are some conditions where it is more common, so people like to have the option to use that. using -ntt 1 most of the time is ok if you only have one dynamic mod. As you add more dynamic modifications, things get slower and slower, and at some point, you just have to use -ntt 2 and only identify fully tryptic peptides (or let MS-GF+ runs for 12 hours, 18 hours, or even several days.