Mouse primary T cell phosphotyrosine proteomics enabled by BOOST

Abstract

AbstractThe Broad Spectrum Optimization of Selective Triggering (BOOST) approach was recently developed to increase the quantitative depth of the tyrosine phosphoproteome by mass spectrometry-based proteomics. While BOOST has been demonstrated in the Jurkat T cell line, it has not been demonstrated in scarce mice primary T cells. Here, we show the first phosphotyrosine proteomics experiment performed in mice primary T cells using BOOST. We identify and precisely quantify more than 2,000 unique pTyr sites from more than 3,000 unique pTyr peptide PSMs using only 1 mg of protein from T cell receptor-stimulated primary T cells from mice. We further reveal the importance of the phase-constrained spectrum deconvolution method (ΦSDM) parameter on Orbitrap instruments that, when disabled, enhances quantitation depth, accuracy, and precision in low-abundance samples. Using samples with contrived ratios, we find that disabling ΦSDM allows for up to a two-fold increase in the number of statistically significant intensity ratios detected while enabling ΦSDM degrades quantitation, especially in low-abundance samples.TOC Graphic