Analysis of Extracellular Vesicle and Contaminant Markers in Blood Derivatives Using Multiple Reaction Monitoring

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

2 Citations (Scopus)

Abstract

Extracellular vesicles (EVs) are naturally occurring membranous particles that can be isolated from blood and other biofluids. EVs have drawn considerable attention for their potential as a minimally invasive biomarker source for a range of conditions, based on tissue-specific expression of proteins and other molecular information. To promote robust characterization of EV isolates, the International Society for Extracellular Vesicles (ISEV) has established consensus minimal requirements for the study of extracellular vesicles (MISEV) reporting guidelines. A core element of MISEV guidance is the recommendation for the analysis of protein markers in samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the sample matrix. Furthermore, there is growing interest in circulating EVs enriched for tissue-specific origin, and in this context, the degree of nontarget EV "contamination" (e.g., EVs derived from blood cells) may inform assessment of sample purity. The increasing application of EVs as a liquid biopsy for clinical applications requires a high-throughput multiplexed approach that enables analysis of protein markers from small volumes of starting material, ideally utilizing the same platform for measuring biomarkers of interest. To this end, targeted liquid chromatography mass spectrometry using multiple reaction monitoring (LC-MRM-MS) is a key platform for the quantitative assessment of target proteins within EV samples. Here we describe a protocol for the isolation of EVs from blood and parallel analytical methods targeting general EV markers and blood cell-derived EV markers, along with guidance of best practice for sample collection and processing.

Original languageEnglish
Title of host publicationSerum/Plasma Proteomics
Subtitle of host publicationMethods in Molecular Biology
EditorsDavid W. Greening, Richard J. Richard J.
Place of PublicationNew York
PublisherSpringer Nature
Chapter20
Pages301-320
Number of pages20
ISBN (Electronic)978-1-0716-2978-9
ISBN (Print)978-1-0716-2977-2
DOIs
Publication statusPublished - 12 Feb 2023

Publication series

NameMethods in molecular biology (Clifton, N.J.)
Volume2628
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • EV characterization
  • Extracellular vesicles
  • Liquid chromatography tandem mass spectrometry
  • Multiple reaction monitoring
  • Plasma
  • Protein markers
  • Serum

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