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High-Throughput Profiling of Proteome and Posttranslational Modifications by 16-Plex TMT Labeling and Mass Spectrometry

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Quantitative Methods in Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2228))

Abstract

Mass spectrometry (MS)-based proteomic profiling of whole proteome and protein posttranslational modifications (PTMs) is a powerful technology to measure the dynamics of proteome with high throughput and deep coverage. The reproducibility of quantification benefits not only from the fascinating developments in high-performance liquid chromatography (LC) and high-resolution MS with enhanced scan rates but also from the invention of multiplexed isotopic labeling strategies, such as the tandem mass tags (TMT). In this chapter, we introduce a 16-plex TMT-LC/LC-MS/MS protocol for proteomic profiling of biological and clinical samples. The protocol includes protein extraction, enzymatic digestion, PTM peptide enrichment, TMT labeling, and two-dimensional reverse-phase liquid chromatography fractionation coupled with tandem mass spectrometry (MS/MS) analysis, followed by computational data processing. In general, more than 10,000 proteins and tens of thousands of PTM sites (e.g., phosphorylation and ubiquitination) can be confidently quantified. This protocol provides a general protein measurement tool, enabling the dissection of protein dysregulation in any biological samples and human diseases.

Kaiwen Yu and Zhen Wang are the co-first authors.

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Acknowledgments

This work was partially supported by the National Institutes of Health (R01GM114260, R01AG047928, R01AG053987, and RF1AG064909) and ALSAC (American Lebanese Syrian Associated Charities). The MS analysis was performed in the Center of Proteomics and Metabolomics at St. Jude Children’s Research Hospital, partially supported by NIH Cancer Center Support Grant (P30CA021765).

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Authors and Affiliations

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

    Kaiwen Yu, Zhen Wang, Zhiping Wu & Junmin Peng

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

    Haiyan Tan, Ashutosh Mishra & Junmin Peng

Authors
  1. Kaiwen Yu
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  2. Zhen Wang
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  3. Zhiping Wu
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  4. Haiyan Tan
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  5. Ashutosh Mishra
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  6. Junmin Peng
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Corresponding author

Correspondence to Junmin Peng .

Editor information

Editors and Affiliations

  1. Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-Universität Bochum, Bochum, Germany

    Katrin Marcus

  2. Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany

    Martin Eisenacher

  3. Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum, Bochum, Germany

    Barbara Sitek

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Yu, K., Wang, Z., Wu, Z., Tan, H., Mishra, A., Peng, J. (2021). High-Throughput Profiling of Proteome and Posttranslational Modifications by 16-Plex TMT Labeling and Mass Spectrometry. In: Marcus, K., Eisenacher, M., Sitek, B. (eds) Quantitative Methods in Proteomics. Methods in Molecular Biology, vol 2228. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1024-4_15

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  • DOI: https://doi.org/10.1007/978-1-0716-1024-4_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1023-7

  • Online ISBN: 978-1-0716-1024-4

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