A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome - Université Paris-Est-Créteil-Val-de-Marne Access content directly
Journal Articles Journal of Lipid Research Year : 2020

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

Abstract

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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Dates and versions

hal-03086102 , version 1 (22-12-2020)
hal-03086102 , version 2 (02-02-2021)

Identifiers

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Cerina Chhuon, Shao-Yu Zhang, Vincent Jung, Daniel Lewandowski, Joanna Lipecka, et al.. A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome. Journal of Lipid Research, 2020, 61 (11), pp.1512-1523. ⟨10.1194/jlr.D120000672⟩. ⟨hal-03086102v1⟩
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