Open search on non-Tryptic peptides

[WrennPockTwo]

Dear FragPipe team,
thanks a lot for creating and maintaining this wonderful tool for mass spectrometry!

My problem is based on the open search. I am used to get unmodified peptides as the major hit in my open search under normal (tryptic) conditions. However, an open search using lysn_promisc as the enzyme does not yield any unmodified peptides. Is this expected or to I use the workflow incorrectly.

Thanks a lot for any help here
Best regards,
Max

I attached the lysn_promisc open search log file: log_2024-01-16_12-31-15.zip and the corresponding output
log_2024-01-16_12-31-15.zip
log_2024-01-16_12-31-15_ptm-shepherd-output.zip

I attached the trypsin open search log file: log_2024-01-04_13-06-20.zip and the corresponding output
log_2024-01-04_13-06-20.zip
log_2024-01-04_13-06-20_ptm-shepherd-output.zip

[danielgeiszler]

Hi Wrenn. Can you please share your psm.tsv files?

It appears that some of your files have parameters that don't match the experimental conditions, namely RA2, RA3, and RA4.

These are passing either no PSMs or invalid delta masses to PTM-Shepherd. Can you confirm the validity of these three PSM tables?

[danielgeiszler]

@fcyu I think this is actually an issue with Philosopher's delta mass column getting mangled, so I'll assign you too since I'm not sure who is in charge of Philosopher at the moment.

[fcyu]

Hi @WrennPockTwo Could share the psm.tsv files?

Thanks,

Fengchao

[WrennPockTwo]

Hi,
thanks for the swift response.
indeed the samples RA2, RA3, and RA4 were empty in the MS run. I have attached the psm.tsv files here:

psm_tsv.zip

I re run my data with only 3 samples to create a smaller dataset with no missing values and indeed the open search does produce the None modification as the top hit. The log, ptm-shepherd output and psm.tsv files attached below. So I guess I have a work around.

github_upload.zip

Cheers,
Max

[danielgeiszler]

Thanks for the files, Max.

@fcyu I think this is the same bug as I've had before. To reproduce this to fix it in Philosopher, you can run a mass offset search that doesn't include 0 and turn off Crystal-C.

[fcyu]

I don't get it. The mass offset only has 0 in the log file. What is wrong with the delta mass column? Can you share a screenshot where you think is wrong?

Thanks,

Fengchao

[danielgeiszler]

Sorry, I thought this was being caused by a previously reported bug in Philosopher. It's a different issue. I'm looking into it.

[fcyu]

@danielgeiszler Is this one fixed?

Thanks,

Fengchao

[danielgeiszler]

Not yet, no.