Lib2HTML truncates peptide sequences after modification

[Juergen Bartel]

Dear all,

I have recently generated an in-silico spectral library for several peptides which may contains different modifications on the same cystein (cystein converted to a serin, formylglycin-aldehyde, ...). For this prediction I used Seq2MS (https://pubs.acs.org/doi/10.1021/acs.jproteome.3c00180) and obtained a library in msp format.

In this file the header indicates modifications for examle in the following way:

Name: DILTPELDNLAQNGSIFTSAYVAHPFCGPSR/2_1(26,C,FGAldehyd)
Comment: Charge=2 Parent=1657.310386 Mods=1(26,C,FGAldehyd) Protein=nan

If I correctly specify the modifications in a .usermods file(*) and import it to spectraST, the resulting sptxt files contain header such as:

Name: DILTPELDNLAQNGSIFTSAYVAHPFC[85]GPSR/2
LibID: 6
MW: 3316.6353
PrecursorMZ: 1658.3177
Status: Normal
FullName: X.DILTPELDNLAQNGSIFTSAYVAHPFC[85]GPSR.X/2 (CID)
Comment: AvePrecursorMz=1659.3531 BinaryFileOffset=19516 Charge=2 FracUnassigned=0.89,4/5;0.83,16/20;0.56,35/60 Mods=1(26,C,FGAldehyd) NAA=31 NISTProtein=nan NMC=0 NTT=1 Parent=1657.310386 Prob=1.0000 Protein=1/nan

However, when I want to check the spectra in this file in HTML format using Lib2HTML, the table contains the complete peptide only for the unmodified form and has an identical truncated peptide for all other modified forms:

Ths does not only affect the truncated amino acids but also results in mis-alignment of the y-ion-series (i.e. due to the modified cystein, y1 is annotated as 122.027 while in this case it should be y5 with a much larger m/z). Again, the annotation in sptxt seems good.

Does anyone has an idea how to solve this and/or how I could watch the spectra alternatively?

Best,

Juergen

(*) part of the usermods file:

C[se]|-15.977156|Serin
C[al]|-17.992806|FGAldehyd

...

[Juergen Bartel]

Dear David,

to generate my library I have done a couple of steps:

1) modified the file utils.py from seq2ms to contain my modifications:

# C H N O S P
# mods = {"ox" : [0,0,0,1,0,0], ## changed this line to enable sulfatase modification prediction se=serine; al=formylglycine-aldehyd; do=formylglycine-diol; ds=diol-sulfate; ss=serine-sulfate
mods = {"ox" : [0,0,0,1,0,0], "se" : [0,0,1,0,-1,0], "al" : [0,-2,1,0,-1,0], "do" : [0,0,2,0,-1,0], "ds" : [0,-1,5,0,0,0], "ss" : [0,0,4,0,0,0],
        "ph" : [0,1,0,3,0,1],
        "cam" : [2,3,1,1,0,0] , "ac": [2,2,0,1,0,0], "me": [1,2,0,0,0,0], "hy": [0,0,0,1,0,0], "gly": [4,6,2,2,0,0],
        "bi" : [10,14,2,2,1,0], "cr": [4,4,0,1,0,0], "di": [2,4,0,0,0,0], "ma": [3,2,0,3,0,0], "ni": [0,-1,1,2,0,0],
        "bu" : [4,6,0,1,0,0], "fo": [1,0,0,1,0,0], "glu": [5,6,0,3,0,0], "hyb": [4,6,0,2,0,0], "pr": [3,4,0,1,0,0],
        "su" : [4,4,0,3,0,0], "tr": [3,6,0,0,0,0], "ci": [0,-1,-1,1,0,0]}

2) I run the prediction by executing 'predict.py' with the a modified input.tsv and (I think) the pretrained_model. The input file looked like the following but contained more peptides:

Sequence Charge Mass Modified sequence Modification Protein
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 2 3332.613578 _DILTPELDNLAQNGSIFTSAYVAHPFCGPSR_ Unmodified
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 3 3332.613576 _DILTPELDNLAQNGSIFTSAYVAHPFCGPSR_ Unmodified
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 4 3332.613576 _DILTPELDNLAQNGSIFTSAYVAHPFCGPSR_ Unmodified
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 2 3316.636422 _DILTPELDNLAQNGSIFTSAYVAHPFC(se)GPSR_ Serine (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 3 3316.636422 _DILTPELDNLAQNGSIFTSAYVAHPFC(se)GPSR_ Serine (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 4 3316.63642 _DILTPELDNLAQNGSIFTSAYVAHPFC(se)GPSR_ Serine (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 2 3314.620772 _DILTPELDNLAQNGSIFTSAYVAHPFC(al)GPSR_ FGAldehyd (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 3 3314.62077 _DILTPELDNLAQNGSIFTSAYVAHPFC(al)GPSR_ FGAldehyd (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 4 3314.620772 _DILTPELDNLAQNGSIFTSAYVAHPFC(al)GPSR_ FGAldehyd (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 2 3332.631336 _DILTPELDNLAQNGSIFTSAYVAHPFC(do)GPSR_ FGDiol (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 3 3332.631336 _DILTPELDNLAQNGSIFTSAYVAHPFC(do)GPSR_ FGDiol (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 4 3332.631336 _DILTPELDNLAQNGSIFTSAYVAHPFC(do)GPSR_ FGDiol (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 2 3411.580326 _DILTPELDNLAQNGSIFTSAYVAHPFC(ds)GPSR_ DiolSulfat (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 3 3411.580326 _DILTPELDNLAQNGSIFTSAYVAHPFC(ds)GPSR_ DiolSulfat (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 4 3411.580324 _DILTPELDNLAQNGSIFTSAYVAHPFC(ds)GPSR_ DiolSulfat (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 2 3396.593236 _DILTPELDNLAQNGSIFTSAYVAHPFC(ss)GPSR_ SerinSulfat (C)
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 3 3396.593235 _DILTPELDNLAQNGSIFTSAYVAHPFC(ss)GPSR_ Unmodified
DILTPELDNLAQNGSIFTSAYVAHPFCGPSR 4 3396.593236 _DILTPELDNLAQNGSIFTSAYVAHPFC(ss)GPSR_ Unmodified

3) I imported the generated .msp file into spectraST format using the command line and the following options:

C:\TPP\bin\spectrast.exe -cNPredicted_Sec2MS.splib -MSec2MS.usermods Predicted.msp

4) I tried to visualise this library by Lib2HTML from the within Petunia

For the files I will send you a link via the ISB contact form to our university's nextcloud where I have uploaded them.

Best regards,

Juergen

[David Shteynberg]

Hello Juergen,

Thanks for providing the test data.  I was able to modify the code to allow Lib2HTML to also read the user modifications and display the from the HTML page.  The prerelease installer that has this feature is available here:  

https://sourceforge.net/projects/sashimi/files/Trans-Proteomic%20Pipeline%20%28TPP%29/TPP%20v7.0%20%28Arafel%29%20rev%20X/TPP_Setup_7.0.x.exe/download

Let me know if you have any other questions.

Cheers!

-David

T

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[Juergen Bartel]

Hi David,

Thank you for modifying the code so quickly! I have just tested Lib2HTML with the new prerelease and it works perfectly fine now.

Cheers!

Juergen

[David Shteynberg]

Excellent! Thank you for letting me know.

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