Background: Cells of the vascular system release spherical vesicles, called microparticles, in the size range of 0.1-1μm induced by a variety of stress factors resulting in variable concentrations between health and disease. Furthermore, microparticles have intercellular communication/signaling properties and interfere with inflammation and coagulation pathways. Today’s most used analytical technology for mecroparticle characterization, flow cytometry, is lacking sensitivity and specificity, which might have led to the publication of contradicting results in the past. Objectives: We propose the use of nano-liquid chromatography two-stage mass spectrometry as a non-biased tool for quantitative MP proteome analysis. Methods: We developed an improved microparticle isolation protocol and quantified the microparticle protein composition of twelve healthy volunteers with a label-free, data-dependent and independent proteomics approach on a quadrupole orbitrap instrument. Results and Conclusions: Using aliquots of 250 μL platelet-free plasma from one individual donor, we achieved excellent reproducibility with an inter-assay coefficient of variation of 2.7 ± 1.7% (mean ± 1 standard deviation) on individual peptide intensities across 27 acquisitions performed over a period of 3.5 months. We show that the microparticle proteome between twelve healthy volunteers were remarkably similar, and that it is clearly distinguishable from whole cell and platelet lysates. We propose the use of the proteome profile shown in this work as a quality criterion for microparticle purity in proteomics studies. We show that multiplexed data-independent acquisition can be used for relative quantification of target proteins using Skyline software.

The data-independent acquisition (DIA) data was acquired with 1 full MS scan from 390 to 1010 m/z at resolution followed by 10 MS2 scans, isolating three randomly combined 10 m/z ion packages (MSX3) covering the mass range from 400 to 1000 m/z, followed by another full MS scan. MaxQuant peptide identifications at 1% FDR were used to build a spectrum library within Skyline. The protein intensities of 78 target proteins were extracted from the DIA MSX3 data using the three most intense peptides on MS1 and MS2 level. For MS2 intensities, 5 to 10 fragment ions were considered. Correctness of extracted chromatographic peaks were evaluated by mProphet (1% FDR) followed by manual validation.

Preparation of platelet-free plasma (PFP) by two different centrifugation steps. Isolation of MP by centrifugation at 16'000g followed by three washing steps with PBS. Lysis of MP in urea buffer followed by a two steps of protein digestion using LysC and trypsin. Shotgun analysis by nanoLC-MS/MS using three replicate injections of each sample on a QExactive Orbitrap instrument.