Expanding TMTpro reagents to 32-plex for high-throughput quantitative proteomics on Orbitrap platforms

Importance of the Topic

Isobaric mass tags have revolutionized multiplexed quantitative proteomics by enabling simultaneous analysis of many samples in a single LC–MS/MS run. Expanding the sample throughput from 18-plex to 32-plex greatly accelerates large-cohort studies, improves statistical power, and reduces instrument time and cost per sample.

Objectives and Study Overview

This work describes the development of a deuterated TMTpro reagent set that adds 17 new reporter ion masses separated by 3 mDa, enabling up to 35-plex analysis on Orbitrap platforms. The primary goal was to characterize the chemical synthesis, labeling efficiency, chromatographic behavior, and quantitative performance of the new 32-plex configuration in comparison with existing 16- and 18-plex formats.

Methodology and Instrumentation

Peptides from HeLa cell digests and protein standards were labeled with traditional and deuterated TMTpro tags using a Thermo Scientific EasyPep workflow. Labeled samples were analyzed on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer coupled to a Vanquish Neo UHPLC with a 50 cm C18 EASY-Spray column. Data were acquired using high-resolution MS1 at 120 K (200 m/z) and HCD FT-MS2 at RP ≥ 75 K or TurboTMT 45 K, as well as RTS SPS-MS3 experiments. Data processing employed Proteome Discoverer 3.0 with SEQUEST HT, UniProt human database, 1% FDR, and reporter-ion integration within an 11 ppm window.

Main Results and Discussion

• Deuterated reporters achieved ~98% deuterium incorporation and labeling efficiency ≥ 99.5% at peptide N-termini and lysine residues.
• Slight retention time shifts of deuterated channels were observed; separate referencing to a matching control channel corrects biases.
• Reporter ions spaced by 3 mDa were baseline-resolved at RP ≥ 75 K (200 m/z) and TurboTMT 45 K, with CVs of 6.4–7.7% within each 16-plex subset and 17% across sets.
• Quantitative performance of 32-plex HCD FT-MS2 matched that of 16-plex and 18-plex formats.
• In RTS SPS-MS3 mode, a single 32-plex run quantified ~15% more proteins and ~39% more peptides than two bridged 18-plex experiments.

Benefits and Practical Applications

• High-throughput profiling of up to 32 samples in one LC–MS/MS run reduces missing data and experimental variability.
• Cost and time savings support large-scale biomarker discovery, drug screening, and clinical proteomics workflows.
• Compatibility with existing TMTpro workflows facilitates easy adoption without significant protocol changes.

Future Trends and Applications

• Extension to full 35-plex workflows combining all reporter sets.
• Integration with data-independent acquisition and advanced quantification algorithms.
• Application in multi-omic and spatial proteomics to maximize sample throughput.
• Automated normalization strategies for cross-set comparisons in longitudinal studies.

Conclusion

The deuterated TMTpro reagent set successfully expands multiplexing capability to 32-plex on Orbitrap platforms without compromising quantitative accuracy, precision, or proteome coverage. This innovation enables more efficient large-scale proteomic experiments and opens new avenues for high-throughput biological and clinical research.

Used Instrumentation

• Orbitrap Eclipse Tribrid mass spectrometer (Thermo Scientific)
• Vanquish Neo UHPLC system with 50 cm C18 EASY-Spray column (Thermo Scientific)
• EasyPep MS sample preparation kit and SPE cleanup (Thermo Scientific)