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Extensive and Accurate Benchmarking of DIA Acquisition Methods and Software Tools Using a Complex Proteomic Standard
Clarisse Gotti1,2, Florence Roux-Dalvai1,2, Charles Joly-Beauparlant2
1Proteomics Platform, CHU de Québec - Université Laval Research Centre, Québec City, Québec G1V 4G2, Canada.
Journal of Proteome Research
|September 2, 2021
Summary
This study compares 36 data-independent acquisition (DIA) workflows for LC-MS/MS proteomics. It provides guidelines for selecting optimal DIA parameters and software for identifying low-abundance proteins in complex proteomes.
Area of Science:
- Proteomics
- Mass Spectrometry
- Analytical Chemistry
Background:
- Data-independent acquisition (DIA) is popular for broad proteome coverage and quantifying low-abundance proteins using LC-MS/MS.
- Lack of consensus exists regarding optimal DIA acquisition parameters and processing tools for complex proteomic analyses.
Purpose of the Study:
- To conduct a comprehensive comparison of various DIA workflows on Orbitrap instruments.
- To provide guidelines for selecting the most suitable DIA workflow for LC-MS/MS proteomic analyses.
Main Methods:
- Tested 36 DIA workflows on Orbitrap instruments using a standard human protein mixture (UPS1) spiked into an E. coli proteome background at 8 concentrations.
- Evaluated 4 DIA window acquisition schemes and 6 software tools (DIA-NN, DIA-Umpire, OpenSWATH, ScaffoldDIA, Skyline, Spectronaut), with and without DDA spectral libraries.
Main Results:
- Assessed workflows based on protein identification numbers, quantification linearity and reproducibility, sensitivity, and specificity across 28 pairwise comparisons.
- Identified key considerations for choosing DIA workflows based on performance metrics.
Conclusions:
- Summarized major considerations for selecting DIA workflows for LC-MS/MS proteomic analyses.
- Proposed guidelines to aid researchers in choosing the best DIA workflow for their specific needs.
- Deposited raw data and software outputs on ProteomeXchange for further research and development.
