MSUM: NEW on Thermo Scientific Orbitrap Fusion Lumos Tribrid MS

Importance of the Topic

An emerging generation of mass spectrometers combines high acquisition speed, real-time intelligent peak detection, ultra-high resolving power and novel fragmentation techniques to address the growing complexity of proteomics, biopharma and metabolomics workflows. Deeper precursor coverage, improved sequence characterization and precise isotopic discrimination enable more confident identifications, quantitation of subtle modifications and streamlined top-down and bottom-up analyses in life science research.

Objectives and Study Overview

The application note describes the integration and performance evaluation of three major innovations on the Thermo Scientific™ Orbitrap Fusion™ Lumos™ MS platform introduced in 2017:

• Advanced Peak Determination (APD) algorithm for enhanced precursor detection in data-dependent acquisition.
• 1M resolution Orbitrap module for baseline separation of near-isobaric species and improved quantitation in peptide mapping.
• UV photodissociation (UVPD) option for complementary fragmentation in top-down, middle-down and small molecule analyses.

Methodology and Instrumentation

The study employed a Thermo Scientific Orbitrap Fusion Lumos MS equipped with:

• The APD algorithm running in real time to identify monoisotopic peaks and charge states at greater depth in complex spectra.
• An ultra-high resolution 1,000,000-resolving-power Orbitrap analyzer module and matching C-trap assembly for field or factory upgrade.
• A class-1 UVPD laser source (213 nm, 2.5 kHz, >1.2 µJ/pulse) integrated into the dual-pressure linear ion trap, enabling UVPD MS/MS detection by ion trap or Orbitrap.

Main Results and Discussion

• APD increased the number of data-dependent MS/MS events by selecting more low-intensity precursors in HeLa tryptic digests, boosting unique peptide identifications by >25 % and protein identifications by >5 % in half the analysis time.
• On-the-fly charge assignment of unresolved intact proteins was demonstrated for carbonic anhydrase, enabling advanced top-down workflows in a single MS1 transient.
• The 1M resolution module resolved a 0.0095 Da mass difference between native and deamidated peptide GFYPSDIAVEWESNGQPENNYK at <1 ppm accuracy, enhancing extraction chromatograms and preventing quantitation bias from coeluting isotopes.
• UVPD provided distinct fragmentation channels in top-down analysis of intact Rituximab heavy chain, achieving 30 % sequence coverage in a single run versus 26 % by ETD HD, and 43 % when combined. In middle-down workflows it contributed 20 % unique sequence information and unambiguous glycosylation site localization.

Benefits and Practical Applications

The combined capabilities allow laboratories to:

• Increase proteome and proteoform coverage with minimal runtime impact.
• Detect and quantify minute post-translational modifications and variant species in complex biopharma samples.
• Execute comprehensive top-down and middle-down protein characterization with complementary fragmentation modes.
• Resolve near-isobaric metabolites, lipids and peptides for accurate flux analysis and biomarker quantitation.

Future Trends and Potential Applications

Ongoing developments may include:

• Integration of machine-learning-driven acquisition methods building on APD for real-time decision making.
• Wider adoption of sub-ppm high-resolving-power modules in routine quality control of biotherapeutics.
• Expansion of UVPD-enabled workflows to lipidomics and small molecule structure elucidation in drug discovery.
• Hybrid fragmentation strategies coupling UVPD with electron-based dissociation for maximal sequence coverage.

Conclusion

The Orbitrap Fusion Lumos innovations introduced in 2017—APD, 1M resolution and UVPD—address key challenges in life science mass spectrometry by maximizing precursor detection, delivering ultra-high resolution separation and offering unique fragmentation chemistries. Together they facilitate deeper, faster and more reliable protein and small molecule analyses for fundamental research and biopharma quality control.