A semi‐automated workflow for DIA‐based global discovery to pathway‐driven PRM analysis

Abstract Targeted proteomics, which includes parallel reaction monitoring (PRM), is typically utilized for more precise detection and quantitation of key proteins and/or pathways derived from complex discovery proteomics datasets. Initial discovery‐based analysis using data independent acquisition (DIA) can obtain deep proteome coverage with low data missingness while targeted PRM assays can provide additional benefits in further eliminating missing data and optimizing measurement precision. However, PRM method development from bioinformatic predictions can be tedious and time‐consuming because of the DIA output complexity. We address this limitation with a Python script that rapidly generates a PRM method for the TIMS‐TOF platform using DIA data and a user‐defined target list. To evaluate the script, DIA data obtained from HeLa cell lysate (200 ng, 45‐min gradient method) as well as canonical pathway information from Ingenuity Pathway Analysis was utilized to generate a pathway‐driven PRM method. Subsequent PRM analysis of targets within the example pathway, regulation of apoptosis, resulted in improved chromatographic data and enhanced quantitation precision (100% peptides below 10% CV with a median CV of 2.9%, n = 3 technical replicates). The script is freely available at https://github.com/StevensOmicsLab/PRM‐script and provides a framework that can be adapted to multiple DDA/DIA data outputs and instrument‐specific PRM method types.