Deep phosphotyrosine characterisation of primary murine T cells using
Broad Spectrum Optimisation of Selective Triggering
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.