Unusual behaviour in limiting number of peptides per protein | Liyan Chen | 2022-07-12 03:44 | |||||||||
Hi Skyline developers, I am using Skyline version 21.2.0.425 for DIA files and typically add all library peptides into the document. Recently we have decided that our data analysis works just fine with maximum of 10-20 peptides per protein, and additional peptides only work to slow down file import. Thus I setup a blank document with these settings update before adding library peptides to document: I still get more than 20 peptides for some proteins whether I select "Pick peptides matching: Library" or "Pick peptides matching library and filter". The only way I can get the limit of nPeptide/protein enforced is to go to "Refine>Advanced" and tick "Auto-select peptides". This refinement takes some time to run and results in repeating sequences within proteins (but still unique to their respective proteins) being introduced as duplicate peptides, and the rest of the peptides within the top 20 rank going missing. In this example using rank 1 GTYSTTVTGR in the protein APOA_Human, only the first instance of this sequence is present in the document when no limit on nPeptides/protein is applied. Ranks 2-20 are occupied by other peptide sequences, with many more other peptides. At this point I had checked for duplicate/repeated peptides in the document and found none. However, when the limit of 20 peptides is enforced using "Refine", each occurrence of GTYSTTVTGR in the protein is re-ranked and counted as separate peptides. How do I get Skyline to only keep one instance of GTYSTTVTGR as rank 1 and the other peptides ranked 2-20 in the document? Is there also a way to limit the peptides when the library peptides are being added to document, instead of adding all and then removing excess peptides? I'm uploading the document "20220712_template.sky.zip" on the filebox. Best regards, |
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