Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome Using Tandem Mass Tags
Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome Using Tandem Mass Tags
复制标题
使用串联质量标签对动态热休克蛋白质组进行准确、灵敏的定量
DOI：
10.1021/acs.jproteome.9b00704
复制
发表时间：
2020
影响因子：
4.4
通讯作者：
Lewis, Jeffrey A.
中科院分区：
文献类型：
--
作者：
Storey, Aaron J.;Hardman, Rebecca E.;Byrum, Stephanie D.;Mackintosh, Samuel G.;Edmondson, Rick D.;Wahls, Wayne P.;Tackett, Alan J.;Lewis, Jeffrey A.
细胞通过调节蛋白质的丰度和功能来应对环境的干扰和侮辱。然而，大多数研究都集中在RNA丰度的变化上，因为从历史上看，定量转录组学比定量蛋白质组学更容易。现代Orbitrap质谱仪现在提供灵敏和深入的蛋白质组覆盖，不仅可以直接、全面地量化蛋白质丰度，还可以对调节蛋白质活性的翻译后修饰(PTM)进行量化。我们实施并验证了芽期酵母的热休克反应，这是一种串联质量标签(TMT)、三阶段质谱学(MS3)策略，用于测量酵母在九个时间点的热休克反应期间蛋白质组的整体变化。我们报告了碱性pH、超高效液相色谱(UPLC)对胰蛋白酶多肽的分级产生最小冗余的超级分，这是后续LC-MS3深度覆盖和定量的关键要求。我们量化了三个生物重复中的2275个蛋白质，发现差异表达在热休克后90分钟左右达到顶峰(有868个差异表达蛋白质，错误发现率为5%)。该方法的敏感性还使我们能够检测泛素化和磷酸化PTM的相对丰度随时间的变化。值得注意的是，翻译后修饰的多肽的相对定量显示了蛋白质PTM调节热休克反应的显著证据。这些数据表明，TMT-MS3的高精度使样品能够在多肽水平上定量，这可以揭示不同实验条件下蛋白质丰度和调节PTM的重要调节。
Cells respond to environmental perturbations and insults through modulating protein abundance and function. However, the majority of studies have focused on changes in RNA abundance because quantitative transcriptomics has historically been more facile than quantitative proteomics. Modern Orbitrap mass spectrometers now provide sensitive and deep proteome coverage, allowing direct, global quantification of not only protein abundance but also post-translational modifications (PTMs) that regulate protein activity. We implemented and validated using the well-characterized heat shock response of budding yeast, a tandem mass tagging (TMT), triple-stage mass spectrometry (MS3) strategy to measure global changes in the proteome during the yeast heat shock response over nine time points. We report that basic-pH, ultra-high performance liquid chromatography (UPLC) fractionation of tryptic peptides yields superfractions of minimal redundancy, a crucial requirement for deep coverage and quantification by subsequent LC–MS3. We quantified 2275 proteins across three biological replicates and found that differential expression peaked near 90 min following heat shock (with 868 differentially expressed proteins at 5% false discovery rate). The sensitivity of the approach also allowed us to detect changes in the relative abundance of ubiquitination and phosphorylation PTMs over time. Remarkably, relative quantification of post-translationally modified peptides revealed striking evidence of regulation of the heat shock response by protein PTMs. These data demonstrate that the high precision of TMT-MS3enables peptide-level quantification of samples, which can reveal important regulation of protein abundance and regulatory PTMs under various experimental conditions.