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

Importance of the Topic

Isobaric tagging strategies are essential in modern proteomics to compare protein abundances across multiple samples in a single experiment. The development of high-plex reagents enhances throughput, reduces instrument time and sample consumption, and supports large-scale studies in areas such as biomarker discovery and quality control.

Aims and Study Overview

This study evaluates a novel set of deuterated TMTpro reagents that introduce a 3 mDa mass shift, expanding multiplexing from 18 to 32 channels on Orbitrap platforms. The objectives include assessing reporter ion resolution, quantification accuracy, retention time behavior, and overall proteome coverage compared to existing TMTpro reagents.

Methodology and Used Instrumentation

HeLa S3 cell lysates were digested, labeled with 32-plex TMTpro reagents (16 traditional and 16 deuterated isotopologues), and combined at defined ratios. Peptides underwent nanoflow reversed-phase LC on a 50 cm C18 EASY-Spray column with a 2–32 % ACN gradient over 120 min at 300 nL/min. MS acquisition employed:

• Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer
• Thermo Scientific Vanquish Neo UHPLC system
• FT-MS¹ scans at 120K resolving power (200 m/z)
• HCD FT-MS² scans at varying resolving powers (TurboTMT 45K, eFT 75K, up to 120K)
• Data-dependent acquisition with 3 sec cycle and 60 sec dynamic exclusion
• Optional RTS SPS-MS³ for improved quantitation

Raw data were processed in Proteome Discoverer 3.2 using SEQUEST HT and Percolator, with reporter ions integrated within an 11 ppm window.

Main Results and Discussion

Resolution experiments show 3 mDa reporter ions are 5 % separated at 75K and baseline resolved at ≥90K. Quantitative tests demonstrate:

• Equivalent accuracy and precision for 32-plex vs. 16-plex at optimized resolving powers (CVs ~6–8 %).
• Retention time shift of 0.5–1 sec between deuterated and non-deuterated channels, correctable via software normalization.
• 32-plex analyses yield 15 % more quantified proteins and 39 % more peptides than two bridged 18-plex runs, reducing missing values.
• Offline high-pH fractionation doubled proteome coverage with over 6 400 proteins quantified in 32-plex.

Benefits and Practical Applications

The expanded 32-plex TMTpro workflow offers:

• High sample throughput in a single LC-MS/MS run
• Reduced instrument time and reagent cost per sample
• Robust quantification with minimal missing data
• Compatibility with existing Orbitrap platforms and software tools

Future Trends and Opportunities

Advances may include further multiplexing beyond 35 channels, integration of real-time search algorithms, improved normalization methods, and automation of sample preparation. Emerging mass analyzers with higher resolution will enhance separation of closely spaced reporters and support deeper proteome coverage.

Conclusion

The new deuterated TMTpro reagents reliably extend multiplexing to 32 channels on Orbitrap instruments without compromising performance. They deliver equivalent accuracy, improved throughput, and simplified workflows for high-throughput quantitative proteomics.