SpectraST: Missing modifications in decoy free library import in ProteomeDiscoverer

[RS]

Hello All,

I'm trying to create a spectral library from Proteome Discoverer (PD  )result files to re-import into PD  as PD accepts sptxt format libraries.

My workflow was as follows:

ProteomeDiscoverer 2.3 msf  format search result files were converted to a  blib file. The filtered blib file was converted to sp2 format. The sp2 format file was finally converted to sptxt format library by SpectraST.

While importing the sptxt format  file (without adding any decoy) I've seen the amino acid modifications used  like Methionine oxidation and Cysteine carbamidomethylation were not recognized by PD

However if I ADD DECOY to the library, followed by its import to PD , the modifications show up in PD (see attached pics)

This is how the header info. of one such modified amino acid peptide appears in the decoy-free library:

Name: M[147]ATALPPR/2

LibID: 329

MW: 873.4731

PrecursorMZ: 436.7366

Status: Normal

FullName: X.M[147]ATALPPR.X/2 (CID)

Comment: AvePrecursorMz=437.0325 BinaryFileOffset=10637539 FracUnassigned=0.90,4/5;0.89,17/20;0.66,1314/1631 Fullname=X.M[147]ATALPPR.X/2 Prob=1.0000 ScanNum=2539.2539 Spec=Raw

NumPeaks: 1631

Can somebody let me know how to incorporate these modifications so that they can be imported by ProteomeDiscoverer
Image links:

Decoy library

 
NO-Decoy library

Thanks

 

[RS]

and I wonder why  Methionine oxidation  (15.995 Da)   is replaced by M [147], is that an internal code to represent that modification or something got messed up?

[Eric Deutsch]

Yes, M[147] is the TPP notation for oxidized methionine. The total mass of oxidized methionine rounds to 147 Da.

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[RS]

Thanks Eric,
Please let me know if you have any  remedies for the rest of the problems

[Eric Deutsch]

I’ve never tried to import into PD, so I can’t help there. Presumably the ADD DECOY doesn’t change things like M[147], so I also do not understand why PD would recognize M[147] in a library with decoys but not in a library without?

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[RS]

I was trying to debug the problem using a single MS2 format Spectra. Here's the Pastebin link 
However, I'm getting an error message while trying to build the spectrst format library:

C:\TPP\bin>spectrast.exe -cNSingleSpectraMet SingleSpectraMet.MS2

SpectraST started at Wed Jul 08 12:20:28 2020.

Total Run Time = 2 seconds.

SpectraST finished at Wed Jul 08 12:20:30 2020 with 1 error(s):

CREATE: Libraries to be imported have illegal formats. Exiting.

Any help?

Thanks

[Eric Deutsch]

I have never tried this (importing from MS2) myself. So it works for a spectrum with no M[147], but it fails for a spectrum with M[147]?

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[RS]

I've used two modifications, Methionine oxidation and Cysteine Carbamidomethylation. None of the modifications were imported into PD from the DECOY-FREE library.
The unmodified peptides worked just fine.

Thanks

[RS]

Is the "Comment:" field is optional? I've seen modification info is mentioned in the comment section differently in NO_DECOY  and PLUS_DECOY spectral lib

Example , for  different peptides in  NO_DECOY  and   PLUS_DECOY  respctively:. Note the Mods=0,  Mods=1/8,C,Carbamidomethyl  etc. tokens 

Comment: AvePrecursorMz=375.4312 BinaryFileOffset=229 FracUnassigned=0.00,0/5;0.09,4/20;0.25,1061/1512 Fullname=X.KSDVEAIFSK.X/3 NAA=10 Prob=1.0000 ScanNum=13667.13667 Spec=DECOY

Comment: AvePrecursorMz=375.4312 BinaryFileOffset=35971 FracUnassigned=0.00,0/5;0.09,4/20;0.25,1061/1512 Fullname=X.KSDVEAIFSK.X/3 Mods=0 NAA=13 NMC=2 NTT=1 OrigPeptide=X.KSDVEAIFSK.X/3 Pep=Semi-tryp Prob=1.0000 Protein=DECOY_0_Unknown Remark=DECOY_0 ScanNum=13667.13667 Spec=DECOY

Comment: AvePrecursorMz=379.7998 BinaryFileOffset=3603257 FracUnassigned=0.28,1/5;0.53,12/20;0.37,482/779 Fullname=X.IGKPHTVPC[160]K.X/3 Mods=1/8,C,Carbamidomethyl NAA=10 NMC=1 NTT=1 OrigPeptide=X.IGKPHTVPC[160]K.X/3 Pep=Semi-tryp Prob=1.0000 Protein=DECOY_0_Unknown Remark=DECOY_0 ScanNum=21336.21336 Spec=DECOY