A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome
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 p...
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doaj-70974ef33c9140cfa7c2fa386d973c8d2021-04-29T04:39:22ZengElsevierJournal of Lipid Research0022-22752020-11-01611115121523A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteomeCerina Chhuon0Shao-Yu Zhang1Vincent Jung2Daniel Lewandowski3Joanna Lipecka4André Pawlak5Dil Sahali6Mario Ollero7Ida Chiara Guerrera8Proteomic Platform Necker, Structure Fédérative de Recherche SFR Necker US24, Paris, France; Institut Mondor de Recherche Biomédicale, INSERM, U955, Créteil, FranceInstitut Mondor de Recherche Biomédicale, INSERM, U955, Créteil, FranceProteomic Platform Necker, Structure Fédérative de Recherche SFR Necker US24, Paris, FranceCEA/DRF/IBFJ/iRCM/LRTS, Fontenay-aux-Roses Cedex, France; INSERM, UMR1274, Fontenay-aux-Roses Cedex, France; Université Paris-Diderot, Paris, France; Université Paris-Sud, Paris, FranceProteomic Platform Necker, Structure Fédérative de Recherche SFR Necker US24, Paris, FranceInstitut Mondor de Recherche Biomédicale, INSERM, U955, Créteil, FranceInstitut Mondor de Recherche Biomédicale, INSERM, U955, Créteil, France; AP-HP (Assistance Publique des Hôpitaux de Paris), Department of Nephrology and Renal Transplantation, Groupe Hospitalier Henri-Mondor, Créteil, France; Université Paris Est Créteil, Créteil, FranceInstitut Mondor de Recherche Biomédicale, INSERM, U955, Créteil, France; Université Paris Est Créteil, Créteil, France; For correspondence: Mario Ollero; For correspondence: Mario OlleroProteomic Platform Necker, Structure Fédérative de Recherche SFR Necker US24, Paris, FranceThe 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.http://www.sciencedirect.com/science/article/pii/S002222752043737Ximmunologylymphocytesmembranestandem mass spectrometrycell signalingdetergent-free |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Cerina Chhuon Shao-Yu Zhang Vincent Jung Daniel Lewandowski Joanna Lipecka André Pawlak Dil Sahali Mario Ollero Ida Chiara Guerrera |
spellingShingle |
Cerina Chhuon Shao-Yu Zhang Vincent Jung Daniel Lewandowski Joanna Lipecka André Pawlak Dil Sahali Mario Ollero Ida Chiara Guerrera A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome Journal of Lipid Research immunology lymphocytes membranes tandem mass spectrometry cell signaling detergent-free |
author_facet |
Cerina Chhuon Shao-Yu Zhang Vincent Jung Daniel Lewandowski Joanna Lipecka André Pawlak Dil Sahali Mario Ollero Ida Chiara Guerrera |
author_sort |
Cerina Chhuon |
title |
A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome |
title_short |
A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome |
title_full |
A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome |
title_fullStr |
A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome |
title_full_unstemmed |
A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome |
title_sort |
sensitive s-trap-based approach to the analysis of t cell lipid raft proteome |
publisher |
Elsevier |
series |
Journal of Lipid Research |
issn |
0022-2275 |
publishDate |
2020-11-01 |
description |
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. |
topic |
immunology lymphocytes membranes tandem mass spectrometry cell signaling detergent-free |
url |
http://www.sciencedirect.com/science/article/pii/S002222752043737X |
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