Callahan Aurora, Chua Xien Yu, Griffith Alijah A, Hildebrandt Tobias, Fu Guoping, Hu Mengzhou, Wen Renren, Salomon Arthur R
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island, USA.
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island, USA.
Proteomics. 2024 Dec;24(23-24):e2400106. doi: 10.1002/pmic.202400106. Epub 2024 Aug 1.
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.
由于细胞中酪氨酸磷酸化丰度较低，利用基于质谱的蛋白质组学对酪氨酸磷酸化蛋白质组进行测序具有挑战性，而在原代细胞或临床样本等稀缺样本中，这一挑战更加复杂。最近开发了选择性触发广谱优化（BOOST）方法，通过利用串联质量标签（TMT）、酪氨酸磷酸化富集和酪氨酸磷酸化负载载体通道，增加低蛋白输入样本中的磷酸酪氨酸测序。在这里，我们通过对BOOST方法的准确性和精密度进行基准测试，并识别与受体刺激相关的磷酸化蛋白质组中的显著变化，证明了BOOST在T细胞受体（TCR）刺激的原代小鼠T细胞中的可行性。使用1毫克蛋白质输入（约2000万个细胞）和BOOST，我们识别并精确量化了2000多个独特的磷酸酪氨酸位点，而非BOOST对照样本中约有300个独特的磷酸酪氨酸位点。我们表明，虽然使用BOOST方法时重复变异会增加，但BOOST不会损害定量精度或对一式三份测量的肽段确定统计学显著性的能力。使用BOOST对许多重要T细胞信号蛋白上以前未表征的磷酸酪氨酸位点进行了定量，并且我们识别了仅用BOOST才能观察到的新的TCR反应性磷酸酪氨酸位点。最后，我们确定Orbitrap仪器上的相位谱去卷积方法会损害BOOST实验中的磷酸酪氨酸定量。
