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vdemichev / DiaNN

DIA-NN - a universal automated software suite for DIA proteomics data analysis.
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how to use DIANN-generated phosphorylated library for DIA analysis #1135

Open Wenhhao opened 3 months ago

Wenhhao commented 3 months ago

Hi Vadim, I generated a library for phosphoproteomics data analysis using DIA-NN 1.9. However, I’m not sure whether I should do some preprocessing before using the generated report-lib.tsv in a library-base workflow with DIA-NN.

  1. The library report seems to be filtered by PeptidoformQValue, while PGQValue, Ms1ProfileCorr, BestPTMQValue, and PTMSiteConfidence do not seem to be considered. Should I apply additional filtering?
  2. How should I regard the ExcludeFromAssay and decoy entries in the library report?
  3. In my test, I obtained approximately 3600 phosphorylated peptides out of a total of 4700 peptides. Is it acceptable to retain the transitions without monitored PTMs in the library for DIA-NN analysis?

The settings for library generation are shown below, and I used five diaPASEF files as input: --qvalue 0.01 --matrices --gen-spec-lib --predictor --prosit --fasta-search --min-fr-mz 200 --max-fr-mz 1800 --met-excision --min-pep-len 7 --max-pep-len 40 --min-pr-mz 300 --max-pr-mz 1800 --min-pr-charge 1 --max-pr-charge 4 --cut K*,R* --missed-cleavages 1 --unimod4 --var-mods 3 --var-mod UniMod:21,79.966331,STY --mass-acc 15.0 --mass-acc-ms1 15.0 --individual-mass-acc --individual-windows --peptidoforms --reanalyse --relaxed-prot-inf --rt-profiling

Looking forward to your reply.

Best wishes, Wenhao

vdemichev commented 3 months ago

Hi Wenhao,

You can use DIA-based DIA-NN libs straight away, including when analysing phospho data. In fact, we've done a great deal of work to make sure it works well for phospho.

  1. You don't need to. But it's OK to filter the library based on PGQValue, PeptidoformQValue and, in some cases, PTMSiteConfidence.
  2. Decoy entries must be included in the library. ExcludeFromAssay is not relevant for most workflows.
  3. Yes, and it's recommended.

Best, Vadim

Wenhhao commented 3 months ago

Hi Vadim,

Thank you for your detailed response. I now have a better understanding of your perspective on the DIA-NN libraries.

And thanks again for developing this software; it is very helpful.

Best, Wenhao