Improved Identification, Quantification Accuracy, and Workflow Efficiency using a Modified Quadrupole Orbitrap Mass Spectrometer and Tandem Mass Tags (TMT) Approach

Importance of the Topic

Multiplexed isobaric tagging with Tandem Mass Tags (TMT) has become a cornerstone technique in quantitative proteomics, allowing simultaneous analysis of up to 11 samples and delivering precise relative protein abundance measurements. High accuracy and depth are essential for diverse applications ranging from basic research to industrial quality control.

Objectives and Study Overview

This study evaluated enhancements to the Orbitrap Exploris 480 mass spectrometer, combined with the FAIMS Pro interface, to overcome limitations in TMT-MS2 quantification. Key goals included improving peptide and protein identification rates, reducing co-isolation interference, and enhancing overall workflow efficiency in TMT11plex experiments using a standardized yeast digest.

Methodology and Instrumentation

• Sample: Thermo Scientific Pierce TMT11plex yeast digest standard (knockout strains Met6, His4, Ura2, and parental BY4741).
• Chromatography: EASY-nLC 1200 with 50 cm EASY-Spray C18 column; 50 min gradient from 8 to 32% acetonitrile at 45 °C.
• Mass spectrometry: Orbitrap Exploris 480 with and without FAIMS Pro interface (compensation voltages –45, –60, –75 CV); Precursor Fit filter and TurboTMT (ΦSDM) algorithm applied to MS2 reporter ions.
• Data processing: Proteome Discoverer 2.3 with SEQUEST HT search engine; 10 ppm MS1 tolerance, 0.02 Da MS2 tolerance, TMT6plex static modification, 1% FDR.

Main Results and Discussion

• TurboTMT (ΦSDM) increased peptide and protein identifications by approximately 10% without extending measurement time, particularly benefiting gradients below 120 min.
• FAIMS Pro interface reduced precursor co-isolation interference, improving the Interference Free Index and enabling more accurate TMT-MS2 quantification.
• Implementation of the Precursor Fit filter further enhanced isolation specificity, reducing ratio compression in complex background experiments.
• Reproducibility tests using 500 ng yeast digest over multiple replicates showed consistent protein quantification (CV ~5.4%) and interference levels around 65–69%.
• Combined features yielded up to 11% more quantified proteins and 19% improvement in proteome depth while maintaining high quantification precision.

Benefits and Practical Applications

The modified hybrid quadrupole-Orbitrap system, augmented with FAIMS Pro, Precursor Fit, and TurboTMT, delivers:

• Higher throughput and deeper proteome coverage in multiplexed experiments.
• Enhanced quantification accuracy by minimizing ratio compression and co-isolation artifacts.
• Improved workflow efficiency for routine QA/QC and research laboratories.

Future Trends and Opportunities

Anticipated developments include integration of extended TMT plexing (e.g., TMT16plex), higher-speed algorithms for real-time data processing, expanded FAIMS CV strategies, and adaptation to data-independent acquisition (DIA) workflows, further boosting sensitivity and quantitative reliability.

Conclusion

The Orbitrap Exploris 480 mass spectrometer, enhanced by FAIMS Pro, Precursor Fit, and TurboTMT, provides a robust platform for precise, high-throughput TMT-MS2 quantitation, addressing key challenges in multiplexed proteomics and enabling comprehensive, reproducible analyses.

References

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