Deep phosphotyrosine characterisation of primary murine T cells using broad spectrum optimisation of selective triggering.

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.