Multiplexed quantitation of post-translational modified peptides in single cells using triggered MS/MS combined with super heavy tandem mass tags

Recent advances in mass spectrometry now allow unbiased proteome profiling of thousands of proteins from single cells using both label-free and labeling approaches. However, a major limitation of unbiased approaches is missing data, which worsens as the sample size increases. In addition, the reproducible measurement of post-translational modifications (PTMs) at the single cell level, particularly those present at lower stoichiometry than their unmodified counterparts, remains an even greater challenge. To overcome this issue, we developed a targeted strategy that combines tandem mass tag (TMT) multiplexing with SureQuant-based triggered MS/MS using super heavy TMT-labeled peptides that are 9 Da heavier than the TMTpro tags as triggers. To showcase the strength of our approach, we established a method quantifying four PTMs of histone H3 (i.e., K14ac, K27me, K27me3, and K79me) at single cell resolution. We demonstrated the robustness in quantitation compared to conventional approaches of data-dependent acquisition and standard parallel reaction monitoring with analytical throughput of 1,080 single cells per day. Furthermore, we applied this strategy to sorted single cells and revealed cellular heterogeneity in histone PTMs. Overall, we developed the targeted strategy with improved sensitivity and throughput for analyzing PTMs in single cells, which we expect will be broadly applicable to multiple types of PTMs while enabling focused analysis.

近年来，质谱技术的最新进展已可借助无标记与标记定量两种方法，实现对单细胞中数千种蛋白质的无偏蛋白质组分析。然而，无偏分析方法的核心局限之一为数据缺失问题，且该问题会随样本量扩大而愈发严重。此外，在单细胞层面实现翻译后修饰（post-translational modifications, PTMs）的可重复定量，尤其是化学计量比低于对应未修饰形式的修饰类型，仍是更具挑战性的难题。为解决上述问题，本研究开发了一种靶向分析策略：将串联质量标签（tandem mass tag, TMT）多重标记技术，与基于SureQuant的触发式串联质谱（MS/MS）相结合，以比TMTpro标签重9 Da的超重TMT标记肽段作为触发剂。为验证该方法的性能优势，我们建立了一种可在单细胞分辨率下定量组蛋白H3四种修饰（即K14ac、K27me、K27me3及K79me）的分析方法。相较于传统的数据依赖采集与标准平行反应监测方法，本方法的定量稳定性更优，单日分析通量可达1080个单细胞。此外，我们将该策略应用于分选后的单细胞样本，揭示了组蛋白修饰的细胞异质性。综上，本研究开发的靶向分析策略提升了单细胞翻译后修饰分析的灵敏度与通量，预计可广泛适用于多种类型的翻译后修饰，并支持针对性分析。