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Deep phosphotyrosine characterisation of primary murine T cells using Broad Spectrum Optimisation of Selective Triggering
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  • Aurora Callahan,
  • Xien Chua,
  • Ali Griffith,
  • Tobias Hildebrandt,
  • Guoping Fu,
  • Mengzhou Hu,
  • Renren Wen,
  • Arthur Salomon
Aurora Callahan
Brown University
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Xien Chua
Brown University
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Ali Griffith
Brown University
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Tobias Hildebrandt
Brown University
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Guoping Fu
Blood Research Institute
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Mengzhou Hu
Brown University
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Renren Wen
Blood Research Institute
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Arthur Salomon
Brown University

Corresponding Author:as@brown.edu

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Abstract

Sequencing the tyrosine phosphoproteome using MS-based proteomics is challenging due to the low abundance of tyrosine phosphorylation in cells, a challenge compounded in scarce samples like primary cells or clinical samples. The broad-spectrum optimization of selective triggering (BOOST) method was recently developed to increase phosphotyrosine sequencing in low protein input samples by leveraging tandem mass tags (TMT), phosphotyrosine enrichment, and a phosphotyrosine-loaded carrier channel. Here, we demonstrate the viability of BOOST in T cell receptor (TCR)-stimulated primary murine T cells by benchmarking the accuracy and precision of the BOOST method and discerning significant alterations in the phosphoproteome associated with receptor stimulation. Using 1 milligram of protein input (about 20 million cells) and BOOST, we identify and precisely quantify more than 2,000 unique pY sites compared to about 300 unique pY sites in non-BOOST control samples. We show that although replicate variation increases when using the BOOST method, BOOST does not jeopardise quantitative precision or the ability to determine statistical significance for peptides measured in triplicate. Many pY previously uncharacterised sites on important T cell signalling proteins are quantified using BOOST, and we identify new TCR responsive pY sites observable only with BOOST. Finally, we determine that the phase-spectrum deconvolution method on Orbitrap instruments can impair pY quantitation in BOOST experiments.
01 Apr 2024Submitted to PROTEOMICS
04 Apr 2024Submission Checks Completed
04 Apr 2024Assigned to Editor
04 Apr 2024Review(s) Completed, Editorial Evaluation Pending
04 Apr 2024Reviewer(s) Assigned
01 Jun 2024Review(s) Completed, Editorial Evaluation Pending
01 Jun 20241st Revision Received
05 Jun 2024Reviewer(s) Assigned
11 Jul 2024Review(s) Completed, Editorial Evaluation Pending
11 Jul 20242nd Revision Received
12 Jul 2024Editorial Decision: Accept