Poster Presentation Australia and New Zealand Society for Extracellular Vesicles Conference 2023

Characterising platelet-rich and platelet-poor plasma EV sampling bias for proteomic biomarker studies (#98)

Samantha J Emery-Corbin 1 2 , Jumana Yousef 1 2 , Clare Shugg 3 , Vineet Vaibhav 1 2 , Laura F Dagley 1 2 , Andrew I Webb 1 4
  1. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia
  2. Division of Advanced Technology and Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
  3. Haematology Department, Royal Melbourne Hospital, Melbourne, Victoria, Australia
  4. Colonial Foundation for Healthy Ageing Centre, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

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.

  1. Geyer PE, Voytik E, Treit PV, Doll S, Kleinhempel A, Niu L, Müller JB, Buchholtz ML, Bader JM, Teupser D, Holdt LM, Mann M. Plasma Proteome Profiling to detect and avoid sample-related biases in biomarker studies. EMBO Mol Med. 2019 Nov 7;11(11):e10427. doi: 10.15252/emmm.201910427. Epub 2019 Sep 30. PMID: 31566909; PMCID: PMC6835559. Format: