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A semi-automated workflow for DIA-based global discovery to pathway-driven PRM analysis
  • +3
  • Jennifer Guergues,
  • Jessica Wohlfahrt,
  • John Koomen,
  • Jonathan Krieger,
  • Sameer Varma,
  • Stanley Stevens, Jr.
Jennifer Guergues
University of South Florida
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Jessica Wohlfahrt
University of South Florida
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John Koomen
Moffitt Cancer Center
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Jonathan Krieger
Bruker Corporation
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Sameer Varma
University of South Florida
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Stanley Stevens, Jr.
University of South Florida

Corresponding Author:smstevens@usf.edu

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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 generated from HeLa cell lysate (200 ng, 45-minute gradient method) as well as canonical pathway information from Ingenuity Pathway Analysis was utilized to generated 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.
Submitted to PROTEOMICS
16 Apr 2024Assigned to Editor
16 Apr 2024Submission Checks Completed
19 May 2024Review(s) Completed, Editorial Evaluation Pending
06 Jul 2024Review(s) Completed, Editorial Evaluation Pending
06 Jul 20241st Revision Received
23 Jul 2024Editorial Decision: Accept