Quantitative Profiling of Circulating Plasma Microparticle Associated Proteins by DDA and DIA nanoLC-MS2

摘要

Cells of the vascular system release a heterogeneous mixture of proteins, lipids, RNA, and cytoplasm components by way of spherical vesicles, e.g. microparticles (MPs), in the size range of 0.1-1 μm. The formation of MPs is induced by a variety of stress factors creating buds on the cell surface through activation of the underlying cytoskeleton. These buds are then shed and MP released. Variable MP concentrations have been detected between health and disease, and MP show different pro- or anticoagulation and angiogenesis properties. Furthermore, MPs have cell signaling and intercellular communication properties and interfere with inflammation. Therefore, MPs represent a repository of biological processes taking place in the vascular system and evidence is accumulating that they are a sort of paracrine effectors in self-healing. Today's most used analytical technology for MP characterization, flow cytometry, is lacking sensitivity and specificity as it relies on light scattering. The detection efficiency of flow cytometry has been reported to be in the order of 2-3% for typical blood plasma MP of size smaller than 0.5 μm. Mass spectrometry is the method of choice for the comprehensive quantitative analysis of many proteins, hence enables the decryption of the origin, quantitation of altering protein profiles, and total mass of MP. In order to study the MP proteome of many samples, and screen for MP-associated biomarkers, we have developed a highly reproducible and quantitative MP assay based on targeted, multiplexed data-independent nanoLC-MS~2 (MSX-DIA).