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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Akhilesh Pandey  

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD069039 | Pride | 2025-12-15

REPOSITORIES: Pride