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Quantifying Proteome and Protein Modifications in Activated T Cells by Multiplexed Isobaric Labeling Mass Spectrometry

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T-Helper Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2285))

Abstract

The dynamic regulation of protein function by altered protein expression and post-translational modifications (PTMs) is essential for T cell function, but it has remained difficult to systemically quantify such events. Mass spectrometry (MS)-based proteomics has become a mainstream tool for comprehensive profiling of proteome and PTMs, especially with the development of multiplexed isobaric labeling methods, such as tandem mass tag (TMT), coupled with high-resolution two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). Here, we introduce a deep proteomics profiling protocol with an optimized 11-plex TMT-LC/LC-MS/MS platform to quantitate whole proteome, phosphoproteome, acetylome, and methylome in activated T cells. The major steps include preparation of activated T cells, protein extraction and digestion, TMT labeling, basic pH reverse phase LC, modified peptide enrichment, acidic pH reverse phase LC-MS/MS, and computational data processing. Approximately 10,000 proteins, 30,000 phosphosites, 2,000 lysine acetylated sites, and 1,000 lysine methylated sites can be identified and quantified from 1 mg of proteins per sample. Quality control steps are implemented in this protocol, and future development, such as nanoscale 16-plex TMT analysis, is discussed. This multiplexed and robust method provides a powerful tool for dissecting proteomic and PTM signatures in T cells at the systems level, and it is equally suitable for other biological samples, including effector T cell subsets.

Haiyan Tan and Daniel Bastardo Blanco Co-first authors.

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Acknowledgements

The authors acknowledge N. Chapman for editing of the manuscript. This work was partially supported by the National Institutes of Health (AG047928, AG053987, GM114260 to J.P.; AI105887, AI131703, AI140761, AI150514, CA221290, NS064599 to H.C.) and ALSAC (American Lebanese Syrian Associated Charities).

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Authors and Affiliations

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

    Haiyan Tan, Boer Xie, Yuxin Li & Junmin Peng

  2. Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Daniel Bastardo Blanco & Hongbo Chi

  3. Integrated Biomedical Sciences Program, University of Tennessee Health Science Center, Memphis, TN, USA

    Daniel Bastardo Blanco

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

    Zhiping Wu & Junmin Peng

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  1. Haiyan Tan
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  2. Daniel Bastardo Blanco
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  4. Yuxin Li
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  5. Zhiping Wu
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Corresponding authors

Correspondence to Hongbo Chi or Junmin Peng .

Editor information

Editors and Affiliations

  1. Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

    Francesco Annunziato

  2. Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

    Laura Maggi

  3. Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

    Alessio Mazzoni

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Tan, H. et al. (2021). Quantifying Proteome and Protein Modifications in Activated T Cells by Multiplexed Isobaric Labeling Mass Spectrometry. In: Annunziato, F., Maggi, L., Mazzoni, A. (eds) T-Helper Cells. Methods in Molecular Biology, vol 2285. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1311-5_23

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  • DOI: https://doi.org/10.1007/978-1-0716-1311-5_23

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1310-8

  • Online ISBN: 978-1-0716-1311-5

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