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

Significance of the topic

Quantitative proteomics using isobaric tags such as Tandem Mass Tags (TMT) has revolutionized the study of protein abundance across multiple samples. Precise measurement of peptide ratios in large plex experiments (TMT10/11‐plex) demands high resolving power and interference reduction. Enhancements in mass spectrometer hardware and gas‐phase separation techniques can improve quantitation accuracy, proteome coverage, and workflow throughput, which are critical for applications in systems biology, biomarker discovery, and quality control in pharmaceutical research.

Study objectives and overview

This study evaluates a modified quadrupole‐Orbitrap platform (Orbitrap Exploris 480) combined with the FAIMS Pro interface and advanced data‐processing features (TurboTMT powered by the ΦSDM algorithm and Precursor Fit filter) to:

• Increase proteome identification depth and quantitative precision for TMT MS2 workflows.
• Reduce co‐isolation interference and ratio compression.
• Enhance scan acquisition rates for high‐plex TMT experiments.

Methodology and Instrumentation

Thermo Scientific Pierce TMT11plex yeast digest standard (four yeast knockout strains plus reference) was used to benchmark performance. Samples were separated by nano‐LC on an EASY‐nLC 1200 with a 50 cm C18 column and 50 min gradient at 45 °C. The Orbitrap Exploris 480 mass spectrometer was operated with and without FAIMS Pro (CVs at –45/–60/–75 V), employing the following innovations:

• TurboTMT (ΦSDM) to boost MS2 resolution without extending transient times.
• Precursor Fit filter to select cleaner isolation windows.
• FAIMS Pro interface for gas‐phase fractionation and interference reduction.

Main results and discussion

Consistency and precision trials using daily TMT QC runs (500 ng digest, 50 min gradient) showed high reproducibility (average peptide CV < 7%) across channels. TurboTMT increased peptide and protein identifications by ~10–19%, notably on shorter gradients (<120 min). FAIMS Pro combined with Precursor Fit reduced PSM‐level co‐isolation interference by up to 30% and improved the interference‐free index, yielding more accurate quantitation. Overall, the integrated platform achieved deeper proteome coverage and higher identification rates compared to standard MS2 (e.g., >11,000 proteins quantified with low ratio compression).

Benefits and practical applications

The enhanced Orbitrap Exploris 480 workflow offers:

• Improved throughput via faster scan cycles and reduced need for offline fractionation.
• Accurate quantitation in high‐plex TMT experiments for clinical proteomics and biomarker validation.
• Robust performance suitable for routine QC in regulated laboratories.

Future trends and applications

Continued innovations in ion mobility, real‐time scan decision algorithms, and increasing multiplexing capacity (e.g., TMTpro 16‐plex) will further elevate throughput and quantitation fidelity. Integration with AI‐driven data analysis pipelines and cloud‐based workflows promises to streamline large‐scale studies. Development of more sophisticated interference filters and dynamic gas‐phase fractionation strategies may enable single‐cell proteomic quantitation with isobaric tags.

Conclusion

The combination of a modified quadrupole‐Orbitrap mass spectrometer, FAIMS Pro interface, and advanced data processing (TurboTMT, Precursor Fit) significantly boosts identification rates, quantitative accuracy, and efficiency in TMT MS2 workflows. This integrated approach provides a robust platform for high‐plex proteomic studies in research and quality‐control settings.

References

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