I am not an expert on pep xml files, but it looks like the PSMs in the pep xml file are very different from the rows in the "psm.tsv" file.

For instance, here is something from line number 43819 from "psm.tsv" which looks like it is a peptide with the +203.079 modification:

but here is what that looks like in the pep.xml file:

<spectrum_query start_scan="6166" uncalibrated_precursor_neutral_mass="1658.8953" assumed_charge="3" spectrum="200921_SAM_OC107_Elute1_Slice4.6166.6166.3" spectrumNativeID="controllerType=0 controllerNumber=1 scan=6166" end_scan="6166" index="2143" precursor_neutral_mass="1658.8948" retention_time_sec="1314.2492294311523">
<search_result>
<search_hit peptide="PAATKPATTKPMVK" massdiff="203.0830078125" calc_neutral_pep_mass="1455.8118" peptide_next_aa="M" num_missed_cleavages="2" num_tol_term="2" protein_descr="Collagen alpha-3(VI) chain OS=Homo sapiens OX=9606 GN=COL6A3 PE=1 SV=5" num_tot_proteins="1" tot_num_ions="52" hit_rank="1" num_matched_ions="9" protein="sp|P12111|CO6A3_HUMAN" peptide_prev_aa="K" is_rejected="0">
<modification_info modified_peptide="PAATKPATTKPM[147]VK">
<mod_aminoacid_mass mass="147.0354" position="12"/>
</modification_info>
<search_score name="hyperscore" value="18.976"/>
<search_score name="nextscore" value="0.000"/>
<search_score name="expect" value="6.993e-05"/>
<ptm_result localization="" best_score_with_ptm="13.720062" score_without_ptm="18.9769" localization_peptide="PAATKPATTKPMVK" second_best_score_with_ptm="12.419948" ptm_mass="203.07938"/>
</search_hit>
</search_result>
</spectrum_query>

Those aren't remotely similar so I am not sure what could be going wrong, but maybe someone else on this support board might have an idea.
-- Nick

thanks, Nick,

So basically, skyle doesn't support the psm.tsv file to work with? This is actually where I take the PSM from MSFragger.
thanks,

Carlos

This is the answer that I got so far:

In the pep.xml file, the PSMs are from MSFragger and the glycosylation information is in the mass diff not in the variable modification. After Philosopher generates the psm.tsv, there is another tool, PTM-Shepherd, in FragPipe that modifies the psm.tsv to refine and append the glycosylations as a variable modification. I think that is why you see different modifications between pep.xml and psm.tsv files.

BiblioSpec supports a simple tab-separated-value format where you just supply the file name, spectrum number, charge and peptide sequence.
The format is documented on this page:
https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BiblioSpec input and output file formats
The format is called "ssl" which stands for "spectrum sequence list".

In theory, it would be straightforward to convert the psm.tsv file that you have into the ssl format that BiblioSpec can handle. Unfortunately, BiblioSpec is expecting a column with a modified sequence with mass differences in square brackets after the amino acids. There is no column that looks like that in psm.tsv, but I wrote an Excel VBA function which generates that from the contents of the "Peptide" and "Assigned Modifications" columns.

I made a .ssl file from your psm.tsv file which I have attached. You can use this file to create a spectral library.
I made a library from just the Slice4 results since that was the only raw file I had.

It's likely that I made a mistake in my Excel macro, so, let me know if the modifications in the "Slice4.blib" library are on the wrong amino acid positions or anything like that.
-- Nick

Dear Nick,

Thank you very much for your help. I think that your macro is working ok, some missense PSM come from PSM (probably from some wrong settings).

I will work with the .ssl, can I use your macro?

Thanks you,

Carlos