UltraPlex-TMT: Expanding Isobaric Hyperplexing via Orthogonal Protease Cleavage.

Isobaric labeling is widely used in quantitative proteomics for its multiplexing capabilities, but scaling beyond current limits remains a challenge. Here, we introduce UltraPlex-TMT, a streamlined and scalable workflow that integrates orthogonal protease digestion with hyperplex TMT/TMTpro labeling to effectively double sample throughput. UltraPlex-TMT can be readily implemented without custom chemistry or instrumentation. We benchmarked UltraPlex-TMT using lysine- and arginine-specific protease digests of a two-species proteome labeled with TMT11plex and TMT18plex across four subplexes in a proof-of-concept pseudo-58-plex design. MS2 acquisition quantified ∼6,000-7,000 proteins per subplex and ∼9,000 in total, with ∼50% overlap across all conditions, generating a robust core proteome set with high quantitative reproducibility. RTS-MS3 acquisition showed similar coverage trends, albeit with fewer quantified proteins. Despite reduced depth, MS3 data provided higher quantification accuracy, illustrating a trade-off between proteome depth and precision. Gene set enrichment analysis revealed strong biological concordance between MS2 and MS3 data, and in all conditions tested, the use of orthogonal protease digestion did not introduce systematic quantification bias. UltraPlex-TMT offers a flexible foundation for isobaric labeling-based high-throughput proteomics and is poised to benefit from faster acquisition platforms and extended multiplexing chemistries, supporting future studies exceeding 200-plex scale, potentially equivalent to subminute analysis.