MS1-Level Proteome Quantification Platform Allowing Maximally Increased Multiplexity for SILAC and In Vitro Chemical Labeling

Quantitative proteomic platforms based on precursor intensity in mass spectrometry (MS1-level) uniquely support in vivo metabolic labeling with superior quantification accuracy but suffer from limited multiplexity (≤3-plex) and frequent missing quantities. Here we present a new MS1-level quantification platform that allows maximal multiplexing with high quantification accuracy and precision for the given labeling scheme. The platform currently comprises 6-plex in vivo SILAC or in vitro diethylation labeling with a dedicated algorithm and is also expandable to higher multiplexity (e.g., nine-plex for SILAC). For complex samples with broad dynamic ranges such as total cell lysates, our platform performs highly accurately and free of missing quantities. Furthermore, we successfully applied our method to measure protein synthesis rate under heat shock response in human cells by 6-plex pulsed SILAC experiments, demonstrating the unique biological merits of our in vivo platform to disclose translational regulations for cellular response to stress.

基于质谱（MS1级）前体离子强度的定量蛋白质组学平台，可凭借优异的定量准确性为体内代谢标记提供独特的技术支持，但存在多重检测能力受限（≤3重）及频繁出现定量缺失值的缺陷。本文报道一款新型MS1级定量平台，可针对既定标记方案实现最优多重检测能力，同时保障出色的定量准确性与精密度。该平台目前支持6重体内细胞培养氨基酸稳定同位素标记（SILAC）或体外二乙基化标记，并配套专用分析算法，同时具备拓展至更高多重检测水平的潜力（例如SILAC可拓展至9重）。对于具备宽动态范围的复杂样品（如全细胞裂解液），本平台可实现高精度定量且无定量缺失值问题。此外，我们通过6重脉冲SILAC实验，将本方法成功应用于人类细胞热激应答过程中蛋白质合成速率的检测，验证了本体内平台可通过揭示细胞应激应答的翻译调控机制，展现其独特的生物学研究价值。