Relative quantification using TMT11plex on a modified Q Exactive HF mass spectrometer

Significance of the Topic

The ability to quantify protein abundance across multiple biological samples in a single high-resolution LC-MS experiment is essential for understanding complex biological systems. Isobaric tagging with TMT reagents allows simultaneous analysis of up to eleven conditions, providing deep insight into protein regulation and interaction networks. Advances in mass spectrometer design directly impact the sensitivity, throughput and quantitative accuracy of multiplexed experiments.

Objectives and Study Overview

This study benchmarks the novel Q Exactive HF-X mass spectrometer against its predecessor, the Q Exactive HF, for relative quantification using TMT11plex. Key objectives include

• Assessing identification and quantification performance under optimized MS/MS resolution settings
• Evaluating acquisition speed improvements
• Determining sensitivity gains at low sample amounts

Used Instrumentation

• Thermo Scientific Easy-nLC 1200 HPLC system
• Thermo Scientific Easy-Spray PepMap RSLC C18 column (25 cm, 75 μm, 2 μm particle, 100 Å)
• Thermo Scientific Q Exactive HF mass spectrometer
• Thermo Scientific Q Exactive HF-X mass spectrometer with electrodynamic ion funnel

Methodology

HeLa cell lysate was enzymatically digested and labeled with TMT11plex reagents. Equal aliquots from all eleven channels were pooled and separated on a nano flow LC gradient (0.1% formic acid in water to 80% acetonitrile over 120 minutes). Both instruments were operated in data dependent acquisition mode, selecting the top 20 precursors for fragmentation. Full MS scans were acquired at 120 000 resolution, while MS/MS scans used 60 000 resolution on the HF and 45 000 resolution on the HF-X with corresponding maximum injection times of 120 ms and 86 ms. Data were processed in Proteome Discoverer 2.1 using SEQUEST HT, with a 1% false discovery rate and reporter ion quantification tolerance of ±20 ppm.

Main Results and Discussion

Analysis of TMT11plex labeled HeLa digest revealed:

• An 18% increase in quantified peptides and an 11% increase in protein groups on the Q Exactive HF-X compared to Q Exactive HF
• A 25% faster acquisition rate at 45k MS/MS resolution on the HF-X
• Baseline resolution of all TMT11 reporter ions at 45k resolution, ensuring accurate ratio determination
• Enhanced sensitivity, particularly at low sample loads (10–1000 ng), with the HF-X outperforming at higher dilution levels

Benefits and Practical Applications

The improved performance of the Q Exactive HF-X enhances multiplexed proteomics workflows by:

• Increasing depth of coverage for complex samples
• Reducing analysis time per experiment
• Improving quantitative accuracy and reproducibility, including post-translational modification studies
• Enabling reliable measurements in low abundance proteomics applications

Future Trends and Potential Applications

Further developments may include

• Expansion to higher plex reagents and novel isobaric chemistries
• Integration of real-time data analysis and adaptive acquisition strategies
• Enhanced ion optics and detection technologies for even faster scan rates
• Automated workflows for large-scale clinical and biomarker studies

Conclusion

The Q Exactive HF-X mass spectrometer demonstrates significant advantages over the previous HF model for relative quantification with TMT11plex. By combining higher acquisition speed, improved ion transmission and adequate resolution at reduced transient times, the HF-X enables deeper, more accurate and faster multiplexed proteomic analysis.

References

1. Navin Rauniyar and John R Yates III Journal of Proteome Research 2014 13(12) 5293–5309
2. Kall L Canterbury J Weston J Noble W S MacCoss M Nature Methods 2007 4 923–925
3. Rosa Viner Ryan Bomgarden Michael Blank John Rogers Anal Chem Study Increasing Multiplexing from 6 to 10-Plex with Reporter Ion Isotopologues
4. Tabiwang N Arrey Xiaoyue Jiang Eugen Damoc et al Thermo Fisher Scientific Application Note Isobaric Mass Tagging Quantification Using Q Exactive Instruments