Extensive and Accurate Benchmarking of DIA Acquisition Methods and Software Tools Using a Complex Proteomic Standard

Over the past decade, the data-independent
acquisition mode has
gained popularity for broad coverage of complex proteomes by LC-MS/MS
and quantification of low-abundance proteins. However, there is no
consensus in the literature on the best data acquisition parameters
and processing tools to use for this specific application. Here, we
present the most comprehensive comparison of DIA workflows on Orbitrap
instruments published so far in the field of proteomics. Using a standard
human 48 proteins mixture (UPS1–Sigma) at 8 different concentrations
in an E. coli proteome background, we tested
36 workflows including 4 different DIA window acquisition schemes
and 6 different software tools (DIA-NN, DIA-Umpire, OpenSWATH, ScaffoldDIA,
Skyline, and Spectronaut) with or without the use of a DDA spectral
library. On the basis of the number of proteins identified, quantification
linearity and reproducibility, as well as sensitivity and specificity
in 28 pairwise comparisons of different UPS1 concentrations, we summarize
the major considerations and propose guidelines for choosing the DIA
workflow best suited for LC-MS/MS proteomic analyses. Our 96 DIA raw
files and software outputs have been deposited on ProteomeXchange
for testing or developing new DIA processing tools.