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

Methods Mol Biol. 2021:2285:297-317. doi: 10.1007/978-1-0716-1311-5_23.

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.

Keywords: Acetylome; Isobaric labeling; Mass spectrometry; Methylome; Phosphoproteome; Post-translational modifications; Proteomics; T cell; Tandem mass tag.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Animals
  • Chromatography, High Pressure Liquid
  • Chromatography, Reverse-Phase
  • Humans
  • Immunomagnetic Separation
  • Lymphocyte Activation*
  • Methylation
  • Phosphorylation
  • Protein Processing, Post-Translational*
  • Proteins / analysis*
  • Proteome*
  • Proteomics*
  • Research Design
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism*
  • Tandem Mass Spectrometry*
  • Workflow

Substances

  • Proteins
  • Proteome