Differential ultracentrifugation enables deep plasma proteomics through enrichment of extracellular vesicles

Anders H Kverneland, Ole Østergaard, Kristina Bennet Emdal, Inge Marie Svane, Jesper Velgaard Olsen

Abstract

Human plasma is a rich source of biomedical information and biomarkers. However, the enormous dynamic range of plasma proteins limits its accessibility to mass spectrometric (MS) analysis. Here, we show that enrichment of extracellular vesicles (EVs) by ultracentrifugation increases plasma proteome depth by an order of magnitude. With this approach, more than two thousand proteins are routinely and reproducibly quantified by label-free quantification and data independent acquisition (DIA) in single-shot liquid chromatography tandem mass spectrometry runs of less than one hour. We present an optimized plasma proteomics workflow that enables high-throughput with very short chromatographic gradients analyzing hundred samples per day with deep proteome coverage, especially when including a study-specific spectral library generated by repeated injection and gas-phase fractionation of pooled samples. Finally, we test the workflow on clinical biobank samples from malignant melanoma patients in immunotherapy to demonstrate the improved proteome coverage supporting the potential for future biomarker discovery.

Original languageEnglish
Article number2200039
JournalProteomics
Volume23
Issue number7-8
Pages (from-to)e2200039
ISSN1615-9853
DOIs
Publication statusPublished - Apr 2023

Keywords

  • Extracellular Vesicles/metabolism
  • Humans
  • Mass Spectrometry/methods
  • Proteome/analysis
  • Proteomics/methods
  • Ultracentrifugation

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