Legacy Library Generation (diaPASEF)

For most data-independent acquisition (DIA) analysis, a well-represented spectral library is required for precursors, peptide, and protein identifications. Currently, we support library generation with the diapysef package directly from a MaxQuant analysis of several DDA-PASEF runs. Make sure diapysef is installed before you proceed.

The general steps for generating the spectral library are, (i) annotating ion mobility values in the MQ output, (ii) correcting retention time and ion mobility values against iRT peptides, (iii) formatting it to OpenSwath read-able format, (iv) generate the spectral library formats (.TraML, .pqp, .tsv) with OpenSwath.

Annotating Ion Mobility

Generating the library requires the DDA output files from MaxQuant as well as one of the original DDA PASEF files.

• msms.txt

• allPeptides.txt

• evidence.txt

• .d folder (DDA PASEF)

The library generation script can then be called:

python create_library.py --pasefdata data.d --mqout path/to/maxquant_data --irt irt_file.tsv

For details and options of the script, simply type:

python create_library.py --help

Generating Assay Library

After generating the spectral library with diapysef, the tsv file can be imported into OpenSwathAssayGenerator and OpenSwathDecoyGenerator as documented in pqp