High-throughput, in-depth and absolute plasma proteome analysis with data-independent acquisition mass spectrometry for biomarker discovery and insight into molecular mechanism of myelodysplastic syndromes

Mass spectrometry-based plasma proteomics has been approved to be a viable tool which can quantify hundreds of proteins in one single-LC-MS/MS experiment for biomarker discovery and gain insights of the disease mechanisms. Here we developed a new strategy exploiting data-independent acquisition (DIA) MS-based workflow for a high-throughput, in-depth and absolute quantification of plasma proteins without depleting high abundant protein in one single shot experiment. We tested spectral libraries, optimized LC instrumentation and DIA-MS experimental parameters, and developed absolute quantification algorithms. The strategy established here is applied to myelodysplastic syndromes (MDS) disease cohort as a pilot study. Total 769 proteins were absolutely quantified cross 22 clinical patient sample spanning over 6 orders of magnitude with quantification limit of 10-20 ng/ml, and 95 differentially expressed proteins were identified by adjusting p-value<0.05. A 10-protein combinations had excellent ability separating MDS to healthy (AUC=0.997). Functions of metabolomic vulnerabilities, acute inflammatory response, focal adhesion and microenvironment were revealed to be related to MDS.