How to match Chromatogram peak with Protein ID?

Question

How to match Chromatogram peak with Protein ID?

Closed this issue 20 days ago · 7 comments

- Describe the feature:
I have very few positive hits (unique labelled peptide) and I am trying to troubleshoot. It would be helpful if I could see which peaks in the chromatogram were identified. I am unsure which output files contain this information.

Thank you,

Answer 1 · 2024-06-22T18:52:14.000Z

Could you share the log file?

Thanks,

Fengchao

Answer 2 · 2024-06-22T18:55:33.000Z

Ok, here it is. I label, capture, and chemically cleave peptides with variable mod: Cys404 (light) and Cys406 (heavy). There appears to be many peptides in the initial search, but very few PSM and peptides in the final output.
log_2024-06-21_15-29-10.txt

Answer 3 · 2024-06-22T18:56:30.000Z

Final PSMs: 2500, peptides: 2000, proteins: 900

Answer 4 · 2024-06-22T18:59:37.000Z

Your data has low-resolution (ion trap) MS2. I don't think it is a good idea to apply low-resolution MS2 for the phospho-enriched isotopic labelled data:

variable_mod_01 = 15.9949 M 3
variable_mod_02 = 42.0106 [^ 1
variable_mod_03 = 79.96633 STY 2
variable_mod_04 = -17.0265 nQnC 1
variable_mod_10 = 404.1518 C 2
variable_mod_11 = 57.0215 C 2
variable_mod_12 = 406.16415 C 2

Using high-resolution (Orbitrap) MS2 should significantly improve the result.

Best,

Fengchao

Answer 5 · 2024-06-22T19:02:11.000Z

Hmm, OK. The data isn't actually Phospho-enriched, I could remove this parameter, I think I just had it on by chance.

Should I still acquire high res MS2 because of the 404 and 406 mass detection attempt?

Answer 6 · 2024-06-22T19:07:07.000Z

Thanks for the clarification. Then, I think you could remove phospho.

And yes, I suggest you still acquire high-res MS2.

Best,

Fengchao

Answer 7 · 2024-06-22T19:08:13.000Z

Ok, thanks a lot for the rapid feedback. I will consult and try again.