Few peptides are included in report though didn't match digestion rule

[gureann]

Dear FragPipe team,

I'm using FragPipe 18.0 to process some DDA data to generate spectral library, and found some peptides that are not in the digestion rule of stricttrypsin appeared in final library. In further exploration, these peptides might more likely from decoy sequences but not forward ones

Here are some examples

1. Library peptide IHTGSLR is from protein Q9R1X5, and the sequence with +-5 AAs per side is EEGKQIHTGSLRLQRTL. The AA before this peptide is Q but not KR
   • The reversed peptide of it is LSGTHIK and is only included in protein Q7TPM9, and the +-5 AAs seq is IILPRLSGTHIKLWKLC. In this case, the previous AA is R
   • In related pepxml from msfragger, IHTGSLR primarily belongs to rev_sp|Q7TPM9|MTMRA_MOUSE and alternatively belongs to sp|Q9R1X5|MRP5_MOUSE
2. Peptide LDLALSR is from Q810B6, and the +-5 seq is NGDLALDLALSRRLESI
   • With K/R conversion, the reversed peptide SLALDLK is in O09174 and Q5DTH5, and the +-5 seqs are ARGKRSLALDLKRSQGV and ESCWSSLALDLKKSNSA (the former one matched digestion rule)
   • In related pepxml from msfragger, LDLALSR primarily belongs to rev_sp|O09174|AMACR_MOUSE and alternatively belongs to sp|Q810B6|ANFY1_MOUSE

There are 26 peptides like above ones found in the final library which has total 179672 peptides. This case happened in FP 18.0 (with msfragger 3.5, philosopher 4.4.0, easypqp 0.1.30), but not in FP 17.1 (with msfragger 3.4, philosopher 4.1.0, easypqp 0.1.26, and this was ran half year ago). DIA_SpecLib_Quant was loaded in both two versions of FP

The only difference I could observe in pepxml files from two versions is these peptides would have alternative proteins (and they are forward ones) written along with search hits in 18.0 while only decoy proteins as primary in 17.1 I'm wondering if this is caused by my unintentionally changed settings or the processing approaches in the workflow are changed

Best, Ronghui

• Upload your log file (If a log file hasn't been generated, go to the 'Run' tab in FragPipe, click 'Export Log', zip the resulting "log_[date_time].txt" file to avoid truncation, then attach the zipped file by drag & drop here.)

Here are two log files, and I'd like to share other files via email if you want log-FP17.1.txt log-FP18.0.txt

[fcyu]

Same as this one (https://github.com/Nesvilab/FragPipe/issues/718). Will fix in the next release.

Best,

Fengchao

[fcyu]

Fixed. Will be available in the next version.

Best,

Fengchao