Multiplexed Quantitation of Post-Translationally Modified Peptides in Single Cells Using Triggered MS/MS Combined with Super Heavy Tandem Mass Tags

Recent advances in mass spectrometry permit 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 a lower stoichiometry than their unmodified counterparts, poses an even greater challenge. To overcome this limitation, 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 demonstrate the feasibility of our approach, we established a method quantifying four PTMs on the histone H3 protein (i.e., K14ac, K23ac, K27me, K27me3, and K79me) at single-cell resolution. We demonstrated robustness in quantitation compared to conventional approaches of data-dependent acquisition and standard parallel reaction monitoring. Further, we applied this strategy to single cells and revealed cellular heterogeneity in histone PTMs. Overall, we developed a 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.