Profiling of Human Circulating Plasma Microparticle Associated Proteins by DDA and DIA

Cells of the vascular system release spherical vesicles, called microparticles (MP), in the size range of 0.1-1μm induced by a variety of stress factors resulting in variable MP concentrations between health and disease. Furthermore, MP have intercellular communication/signaling properties and interfere with inflammation and coagulation pathways. Today’s most used analytical technology for MP characterization, flow cytometry, is lacking sensitivity and specificity, which might have led to the publication of contradicting results in the past. We propose the use of nano-liquid chromatography coupled to two-stage mass spectrometry as a non-biased tool for quantitative MP proteome analysis. Using aliquots of 250 μL platelet-free plasma (PFP) from one individual donor, we achieved excellent inter-assay CV’s of 2.7 ± 1.7% (mean ± 1 S.D.) on individual peptide intensities across 27 data-dependent nanoLC-MS/MS runs performed over a period of 3.5 months. With quantitative proteomics, we show that MP composition between twelve volunteers were remarkably stable. MP protein composition is clearly distinguishable from whole cell lysates and we propose that this trait should be used as a quality criterion of MP purity. Furthermore, MP were damaged by freezing PFP. The damage was articulated by a loss of cytoplasm proteins, encompassing a specific set of proteins involved in regulating dynamic structures of the cytoskeleton, and thrombin activation leading to MP clotting. On the other hand, plasma membrane protein composition (cell markers) was not affected. Finally, we show that multiplexed data-independent acquisition can be used for relative quantification of target proteins using Skyline software.