GlycoPeptide Search for EThCD data

[huongtran182]

Hello,

I'm using Glyco-O-HCD to search for my HCD-pd-EThCD data, but the psm reporting excel always showed an empty sheet for glycan match, or saying no pair scan.

log_2025-01-21_10-59-36.txt

Here is the log. I was wondering if you have any suggestions for any of the parameters inputs?

Thank you for your help!

[dpolasky]

Hi @huongtran182,
To search HCD-pd-EThcD data with the O-Pair module, please load the "Glyco-O-Pair" workflow as the starting point. The Glyco-O-HCD workflow uses PTM-Shepherd, which is not currently compatible with O-Pair search.

Best,
Dan

[huongtran182]

Hi,

I also tried the Glyco-O-Pair workflow but the psm file still has empty values for glycan matches. It only showed those regular PTMs like amidation, pyroglutamylation, etc. Here is the log:

log_2025-01-21_12-14-49.txt

Thank you for these assistace!

[dpolasky]

The O-Pair workflow log shows that PTM-Shepherd was enabled in that run as well, hence the same result. It looks like it is because the "Assign Glycans with FDR" option was checked on the glyco tab. Can you try again without enabling that option? It is not compatible with O-Pair.

Best,
Dan

[huongtran182]

Hello Dan,

Thank you! Still empty values for glycan search/column. Here is the new log:

log_2025-01-21_12-57-02.txt

[dpolasky]

The search finished with no errors, so the results should be in the psm.tsv. Which column(s) are you looking at in the table? Any glycopeptides that were found should have an entry in the "O-Pair Score" column. However, there are not many total PSMs so it's also possible that no glycopeptides are found (nonspecific search with combinatorial glycans is extremely challenging).

Best,
Dan

[huongtran182]

Hi,

I'm looking at the O-Pair score column. Here is the psm result file:

psm.xlsx

I also run the same sample with stepped HCD method, and use "Glyco-O-HCD" workflow with PTM-Shepherd and the search looks fine, like a lot of glycopeptides were identified. Here is the log for that search for your reference:

log_2025-01-17_21-41-11.txt

psm_sHCD.xlsx

Thus, I think the EThCD data should have glycopeptides...

[dpolasky]

Hmm, there aren't even any PSMs with delta masses (i.e., potential glycans) reported in your EThcD data, so nothing is getting passed to O-Pair to localize. Can you share the interact.pep.xml file from the EThcD run? It's possible there are no scans passing the oxonium filtering criteria in MSFragger (you can disable it by setting "Diagnostic ion minimum intensity" to 0 in the Glyco/labile mods box on the MSFragger tab).

Best,
Dan

[huongtran182]

Sorry for the delay. Here is the interact.pep.xml file from the EThcD run:

interact.pep.xml_EThcD.txt

[dpolasky]

No worries! Yes, this confirms the psm.tsv result - there are no spectra getting identified from the glyco part of the search, only unmodified (or non-glyco modified) peptides. My recommendation would be to run the OPair search in a new folder with the oxonium filter disabled (see above), and/or check that there are HCD spectra with oxonium ions in the raw file.

Best,
Dan