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Early Cancer Biomarker Discovery Using DIA-MS Proteomic Analysis of EVs from Peripheral Blood

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Serum/Plasma Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2628))

Abstract

One of the cornerstones of effective cancer treatment is early diagnosis. In this context, the identification of proteins that can serve as cancer biomarkers in bodily fluids (“liquid biopsies”) has gained attention over the last decade. Plasma and serum fractions of blood are the most commonly investigated sources of potential cancer liquid biopsy biomarkers. However, the high complexity and dynamic range typical of these fluids hinders the sensitivity of protein detection by the most commonly used mass spectrometry technology (data-dependent acquisition mass spectrometry (DDA-MS)). Recently, data-independent acquisition mass spectrometry (DIA-MS) techniques have overcome the limitations of DDA-MS, increasing sensitivity and proteome coverage. In addition to DIA-MS, isolating extracellular vesicles (EVs) can help to increase the depth of serum/plasma proteome coverage by improving the identification of low-abundance proteins which are a potential treasure trove of diagnostic molecules. EVs, the nano-sized membrane-enclosed vesicles present in most bodily fluids, contain proteins which may serve as potential biomarkers for various cancers. Here, we describe a detailed protocol that combines DIA-MS and EV methodologies for discovering and validating early cancer biomarkers using blood serum. The pipeline includes size exclusion chromatography methods to isolate serum-derived extracellular vesicles and subsequent EV sample preparation for liquid chromatography and mass spectrometry analysis. Procedures for spectral library generation by DDA-MS incorporate methods for off-line peptide separation by microflow HPLC with automated fraction concatenation. Analysis of the samples by DIA-MS includes recommended protocols for data processing and statistical methods. This pipeline will provide a guide to discovering and validating EV-associated proteins that can serve as sensitive and specific biomarkers for early cancer detection and other diseases.

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Author information

Authors and Affiliations

  1. Tasmanian School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, TAS, Australia

    Camila Espejo, Bruce Lyons & Gregory M. Woods

  2. Central Science Laboratory, University of Tasmania, Hobart, TAS, Australia

    Richard Wilson

Authors
  1. Camila Espejo
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  2. Bruce Lyons
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  3. Gregory M. Woods
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  4. Richard Wilson
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Corresponding author

Correspondence to Richard Wilson .

Editor information

Editors and Affiliations

  1. Molecular Proteomics, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia

    David W. Greening

  2. Department of Biochemistry and Chemistry, School of Agriculture, Biomedicine and Environment, La Trobe Institute for Molecular Science, Melbourne, VIC, Australia

    Richard J. Simpson

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Espejo, C., Lyons, B., Woods, G.M., Wilson, R. (2023). Early Cancer Biomarker Discovery Using DIA-MS Proteomic Analysis of EVs from Peripheral Blood. In: Greening, D.W., Simpson, R.J. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 2628. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2978-9_9

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  • DOI: https://doi.org/10.1007/978-1-0716-2978-9_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2977-2

  • Online ISBN: 978-1-0716-2978-9

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