Dataset Information

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

ABSTRACT: The development of sensitive technologies for T cell-raft proteomics is highly challenging. We explored an innovative strategy, based on suspension trapping (S-trap) sample preparation. Mouse splenocytes were subjected to negative immunoselection for T cell preparation. Rafts were isolated by a detergent-free method and Optiprep gradient ultracentrifugation. Flotillin-1-, LAT- and cholesterol-rich microdomains were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. We identified 2680 proteins in the raft-rich fraction and established a database of 894 bona fide T cell-raft proteins. We performed a differential analysis on the raft-rich fraction from non-stimulated vs. anti-CD3/CD28 TCR-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other, such as Akt2 and Nck1. For the first time a proteomic analysis is performed on rafts from ex-vivo T cells obtained from individual mice, before and after TCR activation. Our results show an increased specificity and sensitivity of the proposed method.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Spleen, T Cell, Epithelial Cell, Cell Culture

SUBMITTER: Cerina Chhuon

LAB HEAD: Ida Chiara Guerrera

PROVIDER: PXD016476 | Pride | 2020-08-13

REPOSITORIES: Pride

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Publications

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

Chhuon Cerina C Zhang Shao-Yu SY Jung Vincent V Lewandowski Daniel D Lipecka Joanna J Pawlak André A Sahali Dil D Ollero Mario M Guerrera Ida Chiara IC

Journal of lipid research 20200807 11

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 chole ...[more]

PMID: 32769147

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Dataset Information

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

Publications

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

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