Hello,
I'm analyzing a TMT 10plex-labeled protein sample using MSGF+ and have adapted the MSGFPlus_Tryp_DynMetOx_Stat_CysAlk_TMT_6Plex_20ppmParTol.txt parameters to fit my needs, since the specific TMT 10plex parameter file is no longer available. My workflow involves trypsin digestion and HCD fragmentation.
After modifying the parameter file and successfully obtaining output data, I'm unsure how to validate that the output accurately reflects my parameter adjustments. Specifically, the presence of mass shifts such as "+229.163" (presumably TMT reporter ions) not only at peptide ends but also internally or at the C-terminus, alongside "+57.021" modifications.
Could someone advise on how to interpret these modifications in the context of my MSGF+ results? Additionally, is there a way to configure MSGF+ to include an extra column in the output that indicates cleavage sites (e.g., R and K for trypsin)?
I've attached both my result file and the modified parameter file for reference.
PrecursorMassTolerance=20ppm
NumMods=3
# Modifications (see below for examples)
StaticMod=229.1629, , fix, N-term, TMT10plex
StaticMod=229.1629, K, fix, any, TMT10plex
StaticMod=C2H3N1O1, C, fix, any, Carbamidomethyl # Fixed Carbamidomethyl C (alkylation, +57.0215)
Hello, I'm analyzing a TMT 10plex-labeled protein sample using MSGF+ and have adapted the MSGFPlus_Tryp_DynMetOx_Stat_CysAlk_TMT_6Plex_20ppmParTol.txt parameters to fit my needs, since the specific TMT 10plex parameter file is no longer available. My workflow involves trypsin digestion and HCD fragmentation.
After modifying the parameter file and successfully obtaining output data, I'm unsure how to validate that the output accurately reflects my parameter adjustments. Specifically, the presence of mass shifts such as "+229.163" (presumably TMT reporter ions) not only at peptide ends but also internally or at the C-terminus, alongside "+57.021" modifications.
Could someone advise on how to interpret these modifications in the context of my MSGF+ results? Additionally, is there a way to configure MSGF+ to include an extra column in the output that indicates cleavage sites (e.g., R and K for trypsin)?
I've attached both my result file and the modified parameter file for reference.
PrecursorMassTolerance=20ppm NumMods=3
# Modifications (see below for examples) StaticMod=229.1629, , fix, N-term, TMT10plex StaticMod=229.1629, K, fix, any, TMT10plex StaticMod=C2H3N1O1, C, fix, any, Carbamidomethyl # Fixed Carbamidomethyl C (alkylation, +57.0215)
DynamicMod=O1, M, opt, any, Oxidation # Oxidized methionine FragmentationMethodID=3 InstrumentID=1 EnzymeID=1 IsotopeErrorRange=-1,2 NTT=2 IgnoreMetCleavage=0 TDA=1 NumThreads=All MinPepLength=6 MaxPepLength=50 MinCharge=2 MaxCharge=5 NumMatchesPerSpec=1
Thank you for your assistance.