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

## 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.

## Key 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 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.