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