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In-Depth Blood Proteome Profiling by Extensive Fractionation and Multiplexed Quantitative Mass Spectrometry

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Serum/Plasma Proteomics

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

Abstract

Blood in the circulatory system carries information of physiological and pathological status of the human body, so blood proteins are often used as biomarkers for diagnosis, prognosis, and therapy. Human blood proteome can be explored by the latest technologies in mass spectrometry (MS), creating an opportunity of discovering new disease biomarkers. The extreme dynamic range of protein concentrations in blood, however, poses a challenge to detect proteins of low abundance, namely, tissue leakage proteins. Here, we describe a strategy to directly analyze undepleted blood samples by extensive liquid chromatography (LC) fractionation and 18-plex tandem-mass-tag (TMT) mass spectrometry. The proteins in blood specimens (e.g., plasma or serum) are isolated by acetone precipitation and digested into peptides. The resulting peptides are TMT-labeled, separated by basic pH reverse-phase (RP) LC into at least 40 fractions, and analyzed by acidic pH RPLC and high-resolution MS/MS, leading to the quantification of ~3000 unique proteins. Further increase of basic pH RPLC fractions and adjustment of the fraction concatenation strategy can enhance the proteomic coverage (up to ~5000 proteins). Finally, the combination of multiple batches of TMT experiments allows the profiling of hundreds of blood samples. This TMT-MS-based method provides a powerful platform for deep proteome profiling of human blood samples.

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Author notes

  1. Xue Zhang and Huan Sun contributed equally with all other contributors.

Authors and Affiliations

  1. Departments of Structural Biology and Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Xue Zhang, Huan Sun, Zhen Wang, Yingxue Fu & Junmin Peng

  2. Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Suiping Zhou, High A. Anthony & Junmin Peng

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  1. Xue Zhang
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  2. Huan Sun
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  3. Zhen Wang
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  4. Suiping Zhou
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  5. Yingxue Fu
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  6. High A. Anthony
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  7. Junmin Peng
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Corresponding author

Correspondence to Junmin Peng .

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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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Zhang, X. et al. (2023). In-Depth Blood Proteome Profiling by Extensive Fractionation and Multiplexed Quantitative Mass Spectrometry. 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_8

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  • DOI: https://doi.org/10.1007/978-1-0716-2978-9_8

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  • Publisher Name: Humana, New York, NY

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