Labile Search with Variable Modification
xLinkKnight opened this issue · comments
I'm attempting to search data using a modified Labile-ADP-ribosylation workflow. I've altered the workflow by attempting to include phosphorylation (STY) as a variable modification. An error occurs in PTMProphet whenever I include the possibility of phosphorylation.
A snippet of the log:
[INFO:] Wait for threads to finish ...
[INFO:] Reading file interact.pep.xml ...
[INFO:] processed 3000/1047586 spectrum_queries [WARNING:] token collision ... token: S[708], API mod mass: 621.027, CMD mod mass: 541.061
[WARNING:] Illegal PTM-shuffled peptide detected with non-matching mass: LATEFITLASPQGNSTS[167]MVAPSS[628]FAIDPYNRR
Neutral Mass (from pepXML) = 4141.7
Neutral Computed Mass for Evaluation = 4061.73
PPM difference = 19307.6
[INFO:] please check your specified modification masses for precision or adjust MODPREC= parameter ...
[WARNING:] Illegal PTM-shuffled peptide detected with non-matching mass: LATEFITLASPQGNSTS[167]MVAPSSFAID[656]PYNRR
Neutral Mass (from pepXML) = 4141.7
Neutral Computed Mass for Evaluation = 4061.73
PPM difference = 19307.6
[INFO:] please check your specified modification masses for precision or adjust MODPREC= parameter ...
[WARNING:] Illegal PTM-shuffled peptide detected with non-matching mass: LATEFITLASPQGNSTS[167]MVAPSSFAIDPY[704]NRR
Neutral Mass (from pepXML) = 4141.7
Neutral Computed Mass for Evaluation = 4061.73
PPM difference = 19307.6
[INFO:] please check your specified modification masses for precision or adjust MODPREC= parameter ...
[WARNING:] Illegal PTM-shuffled peptide detected with non-matching mass: LATEFITLASPQGNSTS[167]MVAPSSFAIDPYNR[697]R
Neutral Mass (from pepXML) = 4141.7
Neutral Computed Mass for Evaluation = 4061.73
PPM difference = 19307.6
[INFO:] please check your specified modification masses for precision or adjust MODPREC= parameter ...
[WARNING:] Illegal PTM-shuffled peptide detected with non-matching mass: LATEFITLASPQGNSTS[167]MVAPSSFAIDPYNRR[697]
Neutral Mass (from pepXML) = 4141.7
Neutral Computed Mass for Evaluation = 4061.73
PPM difference = 19307.6
[INFO:] please check your specified modification masses for precision or adjust MODPREC= parameter ...
[ERROR:] Scan 4349 ... number of mod combinations doesn't match the number of peptides ...
[INFO:] INCREASE the precision of specified PTM masses OR DECREASE 'MODPREC=' parameter and rerun ... exiting ...
Process 'PTMProphet' finished, exit code: 1
Process returned non-zero exit code, stopping
My suspicion is that there is a conflict with shared residues of the additional phopho mod (STY) and that of the ADPr mod (STYKRDE), yet differing delta masses.
The default cmd line argument for site localization is as shown:
KEEPOLD STATIC FRAGPPMTOL=20 EM=1 KREDSTY:541.06111 MINPROB=0.5
Is there a way to rewrite the argument to include phosphorylation as a possible mod for STY sites? To my knowledge, multiple "labile" mods are not yet supported. Is there a way to allow a labile (hybrid mode) and pure variable mod (nonlabile mode) in the same search which have overlapping reside specificity?
Hi,
Yes, you can specify as many mods as you would like for PTMProphet in the command line for PTMProphet, something like the following:
KREDSTY:541.06111,STY:79.9663
Note that PTMProphet will not consider labile modification loss unless you add the corresponding neutral loss(es) for each modification.
Best,
Dan