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bioRxiv · 10.1101/2022.05.13.491817

Mouse primary T cell phosphotyrosine proteomics enabled by BOOST

Chua, X. Y.·Callahan, K. P.·Griffith, A. A.·Hildebrandt, T.·Fu, G.·Hu, M.·Wen, R.·Salomon, A.
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Abstract

The 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 ({Phi}SDM) 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 {Phi}SDM allows for up to a two-fold increase in the number of statistically significant intensity ratios detected while enabling {Phi}SDM degrades quantitation, especially in low-abundance samples. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/491817v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@4a2742org.highwire.dtl.DTLVardef@57645borg.highwire.dtl.DTLVardef@17dbfacorg.highwire.dtl.DTLVardef@b2df00_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Chua, X. Y., Callahan, K. P., Griffith, A. A., Hildebrandt, T., Fu, G., Hu, M., Wen, R., Salomon, A.. 2022-05-13. Mouse primary T cell phosphotyrosine proteomics enabled by BOOST. https://doi.org/10.1101/2022.05.13.491817

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