Question about my parameter file

[JannikSchneider12]

Hello everyone,

I want to use a parameter file to execute a search that was previously done in Sequest. So I want to "translate" the parameters to MSGF+. However nearly every q-value in the results is around 1.0, so I think I have some issues in my parameters. I would be glad, if someone could have a look.

So these are the original Sequest params:

And this is my current MSGF+ parameter file:

Thanks for your time and help

[FarmGeek4Life]

I'm not familiar with using Sequest; does the Sequest search automatically include the TMT6Plex static modifications?

[alchemistmatt]

The TMT6Plex static mod is definitely the issue, since your SEQUEST settings did not have TMT and that is a static mod. We have several example parameter files available at https://github.com/MSGFPlus/msgfplus/tree/master/docs/ParameterFiles

I started with file MSGFPlus_Tryp_Dyn_MetOx_STYPhos_Stat_CysAlk_20ppmParTol.txt and updated it to match your settings, specifying a ±10 ppm search tolerance and these static and dynamic mods:

StaticMod=C2H3N1O1,             C,   fix, any,         Carbamidomethyl    # Fixed Carbamidomethyl C (alkylation, +57.0215)

DynamicMod=O1,                  M,   opt, any,         Oxidation          # Oxidized methionine (+15.9949)
DynamicMod=HO3P,                STY, opt, any,         Phospho            # Phosphorylation STY
DynamicMod=C2H2O,               *,   opt, Prot-N-term, Acetyl             # Acetylation, Protein N-term
DynamicMod=H-9 C-5 N-1 O-1 S-1, *,   opt, Prot-N-term, Met-loss           # Methionine removal from Protein N-term (-131.040485)
DynamicMod=H-7 C-3 N-1 S-1,     *,   opt, Prot-N-term, Met-loss-Acet      # Methionine removal from Protein N-term, then acetylation (-89.02992)

Note that while methionine removal from the protein N-terminus is a common dynamic mod that many search engines check for, I'm not familiar with removing methionine then adding acetylation (and UniMod does not have a corresponding mod for a loss of 89.0299 Da). Nevertheless, it should work. I added spaces in the empirical formulas for the Met-loss mods for readability.

Here's the link to the customized parameter file: MSGFPlus_Tryp_Dyn_MetOx_STYPhos_ProtNTermAcet_ProtNTermMetLoss_Stat_CysAlk_10ppmParTol.txt

[JannikSchneider12]

Hey,

Thanks for your help! Yes I noticed the static modification after I posted this too and it was really dumb that I didnt see this earlier. This was the main reason and now I am getting more significant identifications than in the original search, as expected

[JannikSchneider12]

Regarding the Met-loss+Acetyl, I found this in unimod, so maybe my mass specification was not precise enough?

[alchemistmatt]

I completely missed Met-loss+Acetyl , yes, that's the interim name for the mod.
Thus, change the mod name from Met-loss-Acet to Met-loss+Acetyl , giving:

DynamicMod=H-7 C-3 N-1 S-1,     *,   opt, Prot-N-term, Met-loss+Acetyl      # Methionine removal from Protein N-term, then acetylation (-89.02992)