Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome Using Tandem Mass Tags

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-MS3 enables
peptide-level quantification of samples, which can reveal important
regulation of protein abundance and regulatory PTMs under various
experimental conditions.

细胞可通过调控蛋白质丰度与功能，响应环境扰动与损伤。然而，既往绝大多数研究仅聚焦于RNA丰度变化，这是因为定量转录组学（quantitative transcriptomics）在历史上较定量蛋白质组学（quantitative proteomics）更易实现。如今，先进的Orbitrap质谱仪（Orbitrap mass spectrometer）可实现高灵敏度、深层次的蛋白质组覆盖，不仅能直接对蛋白质丰度进行全局定量，还可对调控蛋白质活性的翻译后修饰（post-translational modifications, PTMs）进行定量分析。本研究以特征明确的酿酒酵母热激应答为模型，对串联质量标签-三级质谱（tandem mass tagging, TMT; triple-stage mass spectrometry, MS3）策略进行了优化与验证，以此定量分析酿酒酵母热激应答过程中9个时间点的蛋白质组全局变化。本研究发现，对胰蛋白酶酶解肽段进行碱性pH超高效液相色谱（ultra-high performance liquid chromatography, UPLC）分级，可获得低冗余度的超组分，这是后续LC-MS3实现深层次蛋白质组覆盖与定量的关键前提。本研究在3次生物学重复中定量到2275个蛋白质，并发现蛋白质差异表达在热激后约90分钟达到峰值，在5%错误发现率（false discovery rate, FDR）阈值下共鉴定到868个差异表达蛋白质。该方法的高灵敏度还使我们能够检测泛素化（ubiquitination）与磷酸化（phosphorylation）PTMs的相对丰度随时间的变化情况。值得注意的是，对翻译后修饰肽段的相对定量分析，揭示了蛋白质PTMs对热激应答的调控作用这一重要证据。本研究数据表明，TMT-MS3的高精度特性可实现样本的肽级定量分析，从而能够揭示不同实验条件下蛋白质丰度与调控性PTMs的重要调控机制。