High‐throughput, in‐depth and estimated absolute quantification of plasma proteome using data‐independent acquisition/mass spectrometry (“HIAP‐DIA”)

Abstract Mass spectrometry‐based plasma proteomics has been demonstrated to be a useful tool capable of quantifying hundreds of proteins in a single LC‐MS/MS experiment, for biomarker discovery or elucidation of disease mechanisms. We developed a novel data‐independent acquisition (DIA)/MS‐based workflow for high‐throughput, in‐depth and estimated absolute quantification of plasma proteins (termed HIAP‐DIA), without depleting high‐abundant proteins, in a single‐shot experiment. In HIAP‐DIA workflow, we generated an ultra‐deep cumulative undepleted and depleted spectral library which contained 55,157 peptides and 5,328 proteins, optimized column length (50 cm) and gradient (90 min) of liquid chromatography instrumentation, optimized 50 DIA segments with average isolation window 17 Th, and selected reference proteins for estimated absolute quantification of all plasma proteins. A total of 606 proteins were quantified in triplicate, and 427 proteins were quantified with CV <20% in plasma proteome. R‐squared value of overlapped 208 endogenous PQ500 estimated protein amounts from HIAP‐DIA and absolute quantification with internal standards was 0.82, indicating high quantification accuracy of HIAP‐DIA. As a pilot study, the HIAP‐DIA approach described here was applied to a myelodysplastic syndromes (MDS) disease cohort. We achieved absolute quantification of 789 plasma proteins in 22 clinical plasma samples, spanning less than six orders of magnitude with quantification limit 10–20 ng/mL, and discovered 95 differentially expressed proteins providing insights into MDS pathophysiology.