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Updated: Jun 18, 2025
Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells
Published on: October 22, 2012
Deep phosphotyrosine characterisation of primary murine T cells using broad spectrum optimisation of selective
Aurora Callahan1, Xien Yu Chua1, Alijah A Griffith1
1Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island, USA.
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 optimisation 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 mg of protein input (about 20 million cells) and BOOST, we identify and precisely quantify more than 2000 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.
Insights
The BOOST method significantly enhances phosphotyrosine site identification in low-input samples, revealing new T cell signaling insights. This technique improves quantitative precision for tyrosine phosphoproteome sequencing.
Area of Science:
- Proteomics
- Cellular Signaling
- Immunology
Background:
- Tyrosine phosphorylation sequencing is difficult due to low abundance, especially in scarce samples.
- The BOOST method was developed to improve phosphotyrosine sequencing in low-input samples.
Purpose of the Study:
- To demonstrate the viability of the BOOST method in T cell receptor (TCR)-stimulated primary murine T cells.
- To benchmark the accuracy and precision of BOOST and identify TCR-responsive phosphoproteome alterations.
Main Methods:
- Utilized the broad-spectrum optimisation of selective triggering (BOOST) method.
- Employed tandem mass tags (TMT), phosphotyrosine enrichment, and a carrier channel.
- Analyzed 1 mg of protein input from primary murine T cells.
Main Results:
- BOOST identified over 2000 unique pY sites, a significant increase from ~300 in non-BOOST samples.
- BOOST maintained quantitative precision and statistical significance despite increased replicate variation.
- Identified novel TCR-responsive pY sites and previously uncharacterized sites on T cell signaling proteins.
Conclusions:
- BOOST is effective for deep phosphotyrosine profiling in low-input T cell samples.
- The phase-spectrum deconvolution method on Orbitrap instruments may hinder BOOST quantitation.
- BOOST enables discovery of new signaling pathways in T cell activation.
