Journal of Lipid Research (Nov 2020)

A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome

  • Cerina Chhuon,
  • Shao-Yu Zhang,
  • Vincent Jung,
  • Daniel Lewandowski,
  • Joanna Lipecka,
  • André Pawlak,
  • Dil Sahali,
  • Mario Ollero,
  • Ida Chiara Guerrera

Journal volume & issue
Vol. 61, no. 11
pp. 1512 – 1523

Abstract

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The analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for bottom-up lipid raftomics based on suspension-trapping (S-Trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection, and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in flotillin-1, LAT, and cholesterol were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2,680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from nonstimulated versus anti-CD3/CD28 T cell receptor (TCR)-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an S-Trap-based approach for sample preparation increases the specificity and sensitivity of lipid raftomics.

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