Enhancing protein quantification and sample throughput with TMTpro 32-plex reagents and extended supporting features from the Thermo Scientific Orbitrap Ascend MultiOmics Tribrid Mass Spectrometer

Importance of the topic

Multiplexed quantitative proteomics enables simultaneous analysis of many biological samples, providing deep insight into protein expression, post-translational modifications, and complex biological processes. The development of TMTpro 32-plex reagents doubles sample throughput per LC-MS/MS run compared to 16-plex, addressing high-throughput demands in drug discovery, chemoproteomics, and clinical research.

Objectives and overview

This study assesses the performance of the Thermo Scientific Orbitrap Ascend MultiOmics Tribrid mass spectrometer combined with the new TMTpro 32-plex reagent set. It compares MS2 and real-time search (RTS) SPS-MS3 acquisition methods across four Orbitrap resolving power settings (TurboTMT 45k, TurboTMT 60k, eFT 75k, eFT 90k) to optimize protein/peptide identification rates and quantification accuracy.

Materials and methods

HeLa cell digests labeled with TMTpro 32-plex at 1:4 and 1:10 ratios of deuterated versus non-deuterated channels were spiked with a six-protein mix and pooled. Samples were cleaned and separated by nanoflow LC using a 140-minute gradient on a Vanquish Neo UHPLC with a PepMap 50 cm C18 column, loading 1 µg per injection. Orbitrap Ascend analyses employed MS2 and RTS SPS-MS3 scans at the four resolving power settings with optimized injection times. Data were processed in Proteome Discoverer 3.2 using SEQUEST HT, applying 1% FDR and 11 ppm reporter ion tolerance.

Instrumentation Used

• Orbitrap Ascend MultiOmics Tribrid Mass Spectrometer
• Vanquish Neo UHPLC system
• PepMap 50 cm C18 Easy-Spray PepMap Neo column
• Proteome Discoverer 3.2 software with SEQUEST HT

Results and discussion

All four resolving power options successfully resolved the 3 mDa spacing of TMTpro reporter ions, with eFT 90k achieving baseline separation and eFT 75k within 5% of baseline. MS2 with TurboTMT 45k delivered the highest protein and peptide counts, while RTS SPS-MS3 improved quantification accuracy by minimizing co-isolation interference. A single 32-plex run quantified equal or more proteins and peptides than two replicate 16-plex runs, increasing sample throughput by over 50%.

Benefits and practical applications

• Doubling multiplexing capacity per run reduces instrument time and cost.
• High resolving power ensures precise reporter ion quantification.
• RTS SPS-MS3 acquisition minimizes interference for robust quantitative results.
• Applicable to large-scale proteomic studies in pharma, clinical labs, and biomarker discovery.

Future trends and possibilities

Integration of AI-driven acquisition strategies and on-the-fly data analysis could enhance selectivity and throughput. Combining higher-plex TMT reagents with advanced separation methods and ultra-high-field mass analyzers may further expand quantitative depth. Potential applications include single-cell proteomics and longitudinal clinical profiling.

Conclusion

The Orbitrap Ascend Tribrid mass spectrometer, with TurboTMT and eFT resolving power modes, effectively supports TMTpro 32-plex workflows, doubling sample capacity without sacrificing identification or accuracy. RTS SPS-MS3 acquisition further refines quantification, making single-run 32-plex experiments superior to multiple 16-plex analyses for high-throughput proteomics.

References

1. Frost D., Paulo J.A., Gygi S.P., Kuhn K., Pike I., Bomgarden R.D. Expanding TMTpro reagents to 32-plex for high-throughput quantitative proteomics on Orbitrap platforms. ASMS 2024 poster.