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.pdResult File Import Issue wes rogers  2024-06-14 14:12
 

Hi Skyline Team,

When building a library from a .pdResult file, I have noticed a discrepancy between the number of “high confidence” peptides found in PD and the number of peptides imported in. When using a Percolator q-value of 0.01 or 0.05, the number of peptides imported into library is often a fraction of the “high confidence” peptides found by CHIMERYS. I am using Skyline daily 23.1.1.520 and Proteome Discoverer 3.1 with CHIMERYS searches primarily. I have attached a few examples. I would be happy to provide additional info or upload the .pdResult files. Do you mind taking a look at this?

Thank you,

Wes
 
 
Nick Shulman responded:  2024-06-14 14:32
Can you send us your .pdResult and .pdResultDetails files (and any other files you think might be helpful)?

It would probably also be helpful if you could send us your Skyline documents.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including spectral libraries.

Files which are less than 50MB can be attached to these support requests. You can always zip up and upload larger files here:
https://skyline.ms/files.url

It might also be helpful if you could send us the list of high confidence peptides that you are expecting to see and we can try to figure out why they are not being included.

It might also be helpful if you could send us some screenshots or other output of what you are seeing in Chimerys which is different from what you are seeing in Skyline. (I am not familiar with Chimerys so I do not know what to ask for).

Usually discrepancies like this have something to do with PSM-level scores versus peptide-level, or posterior error probabilities versus q-values.

I am not the expert in this area but after we get your files I will try to find someone to figure out what is going on.
-- Nick
 
wes rogers responded:  2024-06-14 18:16
Hi Nick,

Thank you for the quick response. Yes, I just uploaded a zipped file to the specified location, titled, "pdResults_Example.zip" with the .pdResult plus the PD output as .txt files. Also, I put together a PDF that analyzes a short list of peptides. Please let me know if you have any questions or need any additional info. Thanks!

Thank you,

Wes
 
Nick Shulman responded:  2024-06-14 23:58
Thank you for sending those files. The screenshots are very helpful.

It appears that the reason that the peptide "AAALEFLNR" gets excluded from the .blib file is that there are other equally confident peptides such as "MLLVDELR" which matched the same spectrum.

I will have to ask my coworkers to find out whether this is the expected behavior.
I would have expected that checking the "Keep ambiguous matches" in Skyline would have resulted in peptides like this being included, but that is not the way it seems to have been written.

One thing that I see is that if I choose a q-value score threshold which is different number than "0.01", such as "0.008" or "0.012", the peptide "AAALEFLNR" does end up getting included.
When the cutoff is specifically 0.01, BiblioSpec only looks for "high confidence" peptides and all high confident peptides appear to BiblioSpec to be equally good and the first peptide encountered for a particular spectrum will be the one that is kept.
When the cutoff is a different number, BiblioSpec does look at the actual q-values and uses that to decide which peptide to keep when multiple peptides are associated with the same spectrum.
-- Nick
 
wes rogers responded:  2024-06-17 05:28
Hi Nick,

Thanks for looking into this and the very detailed explanation. This makes a lot of sense in regards it selecting one peptide per spectrum. If you use a score other than 0.01, can it add multiple peptides using the same spectrum? Or is it also just selecting one peptide per spectrum? And also, any thoughts on how some of the "less confident" peptides made it into the .blib (like AAAAAAGAGPEMVR) at 0.01?

Thank you,

Wes
 
Nick Shulman responded:  2024-06-17 23:25

For .pdresult files, BiblioSpec is only going to add one peptide per spectrum.

I made a special build of Skyline-daily which fixes this behavior, I think.
You can install this special Skyline-daily from here:
https://proteome.gs.washington.edu/~nicksh/SpecialSkylines/PdResultFix/
Because this is not an official Skyline-daily it has not been signed. Windows will try to prevent you from installing it so you will have to hunt around for the "Run Anyway" button. This special build of Skyline will appear on the Windows Start Menu as "Skyline-daily PdResult Fix"

With this fixed version of Skyline, if you check the "Include ambiguous matches" checkbox, you will get all PSMs that map to the same spectra.
If you uncheck that checkbox you will not get any PSMs that map to the same spectrum as another peptide.
At least, that's the way I think it will work, but I have not really verified that.

Do you have any idea why your software did not think that "AAAAAAGAGPEMVR" was high confidence?

When I look at the data in the .pdresult file, it looks like "AAAAAAGAGPEMVR" should be high confidence.
BiblioSpec uses the confidence values from the "TargetPeptideGroups" table.

Here is the query that BiblioSpec uses, where I've added a WHERE clause for that particular peptide:

SELECT psms.PeptideID, psm_spec.MSnSpectrumInfoSpectrumID, psms.Sequence, peps.Confidence, psms.WorkflowID, wf.FileName, 0, psms.Charge
FROM TargetPsms psms 
JOIN WorkflowInputfiles wf ON psms.SpectrumFileId = wf.FileId 
JOIN TargetPsmsMSnSpectrumInfo psm_spec ON psms.PeptideID = psm_spec.TargetPsmsPeptideID   AND psm_spec.TargetPsmsWorkflowID = psms.WorkflowID 
JOIN TargetPsmsTargetPeptideGroups psm_pep ON psms.PeptideID = psm_pep.TargetPsmsPeptideID 
JOIN TargetPeptideGroups peps ON psm_pep.TargetPeptideGroupsPeptideGroupID = peps.PeptideGroupID 
WHERE psms.Sequence = 'AAAAAAGAGPEMVR';

When I run that query using DB Browser for SQLite, I see the number "3" in the "Confidence" column which means High Confidence.

Do you have any idea what your software might be looking at which is saying that is not a confident match?
-- Nick
 
wes rogers responded:  2024-06-18 16:19

Hi Nick,

Thank you for working on this and providing this special build. I was able to install and test it out today by importing several .pdResult files. I uploaded the results along with some quick analysis to the file share. It is titled, "pdResults_New_Examples.zip". There were only a very small percentage of peptides missing so I think it worked pretty well. I plan to do some more testing but would like to get your thoughts. Do you think this build could be incorporated into a future Skyline release?

Also, you are correct - the "AAAAAAGAGPEMVR" is listed as "High Confidence." My apologies - I didn't see it listed initially because of the way it was sorted but it is there and "High Confidence." Sorry about that!

Thank you,

Wes