Comet2018+PTMProphet

[Janel]

Hi,

First off I'll just say that it's my first time using PTM Prophet and I'm excited to see what it can do! I'm running Comet->peptideprophet->ptmprophet in TPP v 5.2.0 and unfortunately, I'm running into a number of these errors: 

(example)

ERROR: Cannot initialize for sequence: nKVIDGIDPPK[242], unknown mods may exist in spectrum Rep1_wt_010.12231.12231.1

Please specify modification and rerun PTMProphet ...

Here's the command I used for PTMProphet:

EXECUTING: cd c:/Users/.../prophet-testing-HBH/Test && c: && C:/TPP/bin/PTMProphetParser STY:79.966,MW:15.995,K:114,n:42.010565 MINPROB=0.5 MAXTHREADS=0 FRAGPPMTOL=500 c:/Users/.../prophet-testing-HBH/Test/interact.ipro.pep.xml interact.ptm.ipro.pep.xml 

It looks like PTMProphet isn't reading the mass of the modification correctly, but it isn't all the time (i.e. some of the modifications are analyzed even though they are also the total mass of the aa instead of just the mass change). I also tried using high and low mass accuracy files and changing the FRAGPPMTOL, but still got the same errors. I must be missing something, but I'm not sure what. An option? Something else? Any help would be appreciated!

Thanks in advance,

Janel 

[David Shteynberg]

Hello Janel,

Thank you for trying out PTMProphet.   An internal check in the code verifies that the mod mass during the search matches the mod mass at the time of running PTMProphet.   Can you verify the 114 on K is in fact the mass you used at search time?  Can you look at that specific spectrum in the pepxml file and all check modification listed for that search_hit?

Thanks,

David

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[Janel Beckley]

Hi David,

I just needed to have the exact mass for the K mod. Now it works without errors. Thanks!

I have a couple more questions...about file merging in the TPP. If I want to combine a series of MS experiments each with multiple files, I presume I should always run iprophet to combine files prior to running proteinprophet, right? Also, does it matter if PTMprophet is run before or after iprophet? Thanks for all your help!

Best,

Janel

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[David Shteynberg]

Yes, it makes sense to always run iProphet, to combine the results at the peptide level before your combined them at the protein level with ProteinProphet.  Your best bet is to combine your PeptideProphet analyses into a single analysis with iProphet and then run ProteinProphet on one iProphet output.  This may not always be possible given available computer resources.  In this case, you can break your data up into several iProphet analyses and combine these into one ProteinProphet analysis.  It doesn't matter much when you run PTMProphet, but if you run it after iProphet it might run a bit faster since it will only compute the localization of the combined results.

Cheers,

-David

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[Janel Beckley]

Ok, thanks! Just trying to figure out best practices.

Best,

Janel

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