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

Area of Science:

• Proteomics
• Mass Spectrometry
• Quantitative Biology

Background:

• Isobaric labeling techniques like TMT/TMTpro are crucial for multiplexed quantitative proteomics.
• Current methods face challenges in scaling to higher sample throughput.

Purpose of the Study:

• To introduce UltraPlex-TMT, a novel workflow for enhanced sample throughput in quantitative proteomics.
• To demonstrate the scalability and effectiveness of UltraPlex-TMT using hyperplex TMT/TMTpro labeling and orthogonal digestion.

Main Methods:

• Integration of orthogonal protease digestion (lysine- and arginine-specific) with hyperplex TMT/TMTpro labeling.
• Benchmarking using a two-species proteome in a pseudo-58-plex design with TMT11plex and TMT18plex.
• Quantitative analysis using both MS2 and RTS-MS3 acquisition methods.

Main Results:

• UltraPlex-TMT effectively doubled sample throughput without custom chemistry or instrumentation.
• Achieved quantification of ~6,000-7,000 proteins per subplex and ~9,000 total proteins with high reproducibility.
• Demonstrated a trade-off between proteome depth (MS2) and quantification accuracy (MS3), with strong biological concordance between methods.
• Orthogonal digestion did not introduce systematic quantification bias.

Conclusions:

• UltraPlex-TMT provides a flexible and scalable foundation for high-throughput proteomics.
• The workflow supports future advancements towards ultra-high plex scale (200+ samples) and rapid analysis.
• UltraPlex-TMT facilitates robust proteome profiling with high quantitative accuracy and reproducibility.