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.
Mun et al. (Tue,) studied this question.