UltraPlex-TMT: Expanding Isobaric Hyperplexing via Orthogonal Protease Cleavage
Theodoros I. Roumeliotis, Fernando J. Sialana, Jenny Ho, Jyoti S. Choudhary
TL;DR
UltraPlex-TMT is a new method in proteomics that doubles sample throughput by combining isobaric labeling with orthogonal protease digestion.
Contribution
Introduces UltraPlex-TMT, a scalable workflow that doubles sample throughput in isobaric labeling proteomics.
Findings
UltraPlex-TMT achieved quantification of ~9,000 proteins in total across subplexes with high reproducibility.
Orthogonal protease digestion did not introduce systematic quantification bias.
MS3 data showed higher quantification accuracy compared to MS2 despite reduced depth.
Abstract
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…
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Taxonomy
TopicsAdvanced Proteomics Techniques and Applications · Click Chemistry and Applications · Biotin and Related Studies