利用选择性触发的广谱优化技术深入分析原代小鼠 T 细胞的磷酸酪氨酸特征。

IF 3.4 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Proteomics Pub Date : 2024-08-01 DOI:10.1002/pmic.202400106
Aurora Callahan, Xien Yu Chua, Alijah A Griffith, Tobias Hildebrandt, Guoping Fu, Mengzhou Hu, Renren Wen, Arthur R Salomon
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引用次数: 0

摘要

由于细胞中酪氨酸磷酸化的丰度较低,使用基于质谱的蛋白质组学方法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,这一挑战更为严峻。最近开发的广谱优化选择性触发(BOOST)方法利用串联质量标记(TMT)、磷酸酪氨酸富集和磷酸酪氨酸载体通道,提高了低蛋白质输入样本中磷酸酪氨酸测序的效率。在这里,我们通过对 BOOST 方法的准确性和精确性进行基准测试,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,并发现了与受体刺激相关的磷酸化蛋白质组的显著变化。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 pY 位点,而非 BOOST 对照样本中只有约 300 个独特的 pY 位点。我们的研究表明,虽然使用 BOOST 方法会增加重复性差异,但 BOOST 并不影响定量的精确性,也不影响对一式三份测定的肽段进行统计意义判定的能力。使用 BOOST 对重要 T 细胞信号蛋白上许多以前未定性的PY 位点进行了量化,我们还发现了只有使用 BOOST 才能观察到的新的 TCR 响应PY 位点。最后,我们确定 Orbitrap 仪器上的相谱去卷积方法会影响 BOOST 实验中的PY 定量。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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.

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来源期刊
Proteomics
Proteomics 生物-生化研究方法
CiteScore
6.30
自引率
5.90%
发文量
193
审稿时长
3 months
期刊介绍: PROTEOMICS is the premier international source for information on all aspects of applications and technologies, including software, in proteomics and other "omics". The journal includes but is not limited to proteomics, genomics, transcriptomics, metabolomics and lipidomics, and systems biology approaches. Papers describing novel applications of proteomics and integration of multi-omics data and approaches are especially welcome.
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