In plasma proteomics, biomarker discovery is hindered by highly-abundant proteins that obscure clinically-relevant candidates. Among new methods shifting detection limits to low-abundance proteins, plasma-derived, circulating extracellular vesicles (EV) have emerged. However, rigorous standards for plasma-derived EVs are lacking.
We explored EV proteomes of patient-matched platelet-rich plasma (PRP) and platelet-poor plasma (PPP) in a PRP-titration experiment. We tested antibody-based (ExoNet, INOVIQ) and SAX-based (MagNet, ReSyn) reagents, analysing neat plasma alongside EV samples on a timsTOFpro (Bruker) mass spectrometer using diaPASEF. An ‘additive’ platelet-derived contamination was observed in both reagents with >1500 unique, reproducible protein identifications in PRP-EVs as compared to PPP-EVs, and >1000 in titrated PRP-EV samples. Notably, <5 unique identifications in PPP-EVs compared to PRP-EVs highlights the ‘overlapping’ background complicating resolution of platelet-contaminated EV proteomes. Principal component analysis revealed linear separation of titrated PRP in neat plasma, with all PRP-EV samples grouped separately from PPP-EV (PC1: 46.1% ExoNet, 65.2% MagNet, 24.8% neat plasma). This was confirmed by proteins linearly correlated with PRP/PPP. However, our PRP-correlated EV proteins did not overlap with the Platelet Quality Panel (Geyer et al, 2019), with only 1/26 (ExoNet) and 9/26 (MagNet) correlated, emphasizing the potential for an EV-specific contamination panel developed from our dataset.
This work will clarify the contributions of platelet microvesicles in plasma EV biomarker discovery, particularly relative to patient variation. By doing so, we aim to prevent pre-analytical errors, provide novel EV-markers, and ensure accurate quantification and correction of platelet-derived statistical artefacts, paving the way for standardised plasma-derived EV research.