Thermo Scientific Q Exactive HF Orbitrap LC-MS/MS System

Significance of High-Field Orbitrap MS in Analytical Chemistry

The Q Exactive HF hybrid quadrupole–Orbitrap LC-MS/MS system addresses critical needs in proteomics, metabolomics, lipidomics and biopharmaceutical analysis by combining ultra-high resolution, rapid scan speed and robust quantitative performance. In modern laboratories, where sample throughput, sensitivity and data quality drive discovery and quality control, this platform offers transformative improvements over previous generation instruments.

Objectives and Study Overview

This application note characterizes the performance of the Thermo Scientific Q Exactive HF mass spectrometer, built on the proven Q Exactive Plus platform. Key goals include demonstrating:

• Enhanced scan speed and resolving power via the ultra-high-field Orbitrap analyzer
• Improved precursor selection and transmission through Advanced Quadrupole Technology
• Quantitative workflows using data-independent acquisition (DIA) and parallel reaction monitoring (PRM)
• Capability to analyze intact proteins and complex lipids with high confidence

Methodology and Instrumentation

The system integrates:

• Ultra-High-Field Orbitrap (HF) mass analyzer for faster MS and MS/MS scans at resolutions up to 240,000 (FWHM at m/z 200)
• Advanced Quadrupole Technology (AQT) enabling narrow isolation windows down to 0.4 amu for high selectivity
• Advanced Active Beam Guide (AABG) RF lens to reduce noise and enhance robustness
• Higher-energy collisional dissociation (HCD) cell for efficient fragmentation
• Optional Intact Protein Mode for resolving large proteoforms and antibodies up to 50 kDa

Main Results and Discussion

Proteomics performance:

• 30 % increase in unique peptide identifications and 20 % more protein groups in standard gradients compared to Q Exactive Plus
• 37 % shorter cycle time (1.13 s vs. 1.77 s) at 60,000 resolution with 20 HCD MS/MS scans, enabling steeper gradients and doubled throughput

Quantitative capabilities:

• PRM achieves routine quantitation down to 0.01 fmol peptides with coefficients of variation below 3 % using 0.7 amu isolation
• Narrow quadrupole windows remove interferences, improving accuracy in complex matrices
• Fast scan rates sample narrow UHPLC peaks more densely, preserving peak shape and precision

Data-independent acquisition (DIA):

• Multiple DIA methods (BASIC-DIA, pSMART-DIA, MSx-DIA) create permanent digital archives for retrospective analysis
• High resolution (<3 ppm mass accuracy) reduces false positives and improves selectivity compared with SWATH-type approaches

Intact protein analysis:

• Optional mode resolves isotopic peaks of intact antibodies (∼150 kDa) and subunits, distinguishing glycoforms with 1–10 ppm mass accuracy
• Routine characterization of mAbs and biotherapeutic proteoforms with high resolving power

Lipidomics:

• Identification of over 20 % more lipid species in a bovine heart extract using a 33 % shorter gradient
• Up to 120,000 resolving power required to separate isobaric lysophospholipids, enabling confident assignments

Benefits and Practical Applications

Key advantages for analytical laboratories include:

• High throughput discovery and quantitation across omics disciplines
• Robust performance for challenging samples in QA/QC and regulated environments
• Flexible modes (DDA, DIA, PRM) to balance sensitivity, selectivity and archival needs
• Compatibility with advanced software suites for data processing and reporting (Proteome Discoverer, Pinpoint, PepFinder, Protein Deconvolution, LipidSearch)

Future Trends and Applications

Emerging directions enabled by high-field Orbitrap technology include:

• Deep proteome profiling in single-cell and micro-sample workflows
• Expanded intact proteoform mapping for advanced biotherapeutic characterization
• High-throughput lipidomics and metabolomics for systems biology and clinical research
• Integration with machine learning for automated method optimization and data interpretation

Conclusion

The Q Exactive HF LC-MS/MS system delivers a significant leap in speed, resolution and quantitative accuracy across diverse analytical applications. Its combination of ultra-high-field Orbitrap detection, advanced quadrupole isolation and versatile acquisition modes provides laboratories with the performance necessary to tackle the most demanding discovery and routine workflows.

Reference

No formal literature references were provided in the source application note.