Influence of Ion Storage Location and Multiple Fill/Laser Pulse Sequence on Top-down UVPD Using an Orbitrap Fusion Mass Spektrometer

Importance of the Topic

Ultraviolet photodissociation (UVPD) has emerged as a powerful tool for top-down proteomics by generating extensive sequence-informative fragment ions. Integrating UVPD with high-resolution mass analyzers enhances the characterization of intact proteins and proteoforms, addressing challenges in sequence coverage, charge-state dependence, and product-ion yield.

Objectives and Study Overview

This study evaluates the performance of UVPD on a Thermo Fisher Scientific Orbitrap Fusion Tribrid mass spectrometer. Two key factors are explored: the ion storage location for UVPD (high-pressure cell, low-pressure cell, and ion routing multipole) and the effect of multiple fill/laser-pulse sequences on fragmentation efficiency and sequence coverage.

Methodology

• Model protein: Apomyoglobin (Sigma Aldrich), diluted to 0.5 µM in 49:49:2 water:methanol:acetic acid.
• UVPD source: 193 nm ArF excimer laser (Coherent ExciStar XS), pulse energy 0.4–60 mJ, 5 ns FWHM, integrated via a UV-transparent window flange.
• Ion storage locations tested: low-pressure cell (LPC), high-pressure cell (HPC), and ion routing multipole (IRM/HCD cell).
• Fill/laser-pulse schemes: single fill/single pulse; single fill/multiple pulses; multiple fills/multiple pulses.
• Data acquisition: High-resolution Orbitrap detection of precursor and fragments; non-interrupted laser alignment under vacuum.
• Data analysis: Peak deconvolution with Hardklor; database searching with Prosight Lite.

Main Results and Discussion

• UVPD in the HPC provided more stable and higher overall sequence coverage across a wide energy range compared to the LPC, which exhibited a faster coverage roll-off at energies above 0.6 mJ per pulse.
• At low pulse energy (0.2 mJ), both LPC and HPC achieved extensive fragmentation; at high energy (1.0 mJ), the LPC lost coverage in central sequence regions, consistent with multi-generation fragment pathways.
• Multiple fill and multiple pulse schemes improved fragment ion yield without excessive precursor depletion when optimized; a 5-fill/5-pulse sequence at moderate energy balanced coverage and signal intensity.
• Ion protection via resonant dipolar excitation successfully prevented over-fragmentation by ejecting unreacted precursor out of the laser path.

Benefits and Practical Applications

• Enhanced sequence coverage in top-down proteomics, facilitating identification and localization of post-translational modifications.
• Robust control over fragmentation through tuning of pulse energy, location, and fill/pulse schemes.
• Compatibility with vacuum-integrated laser alignment allows reproducible operation in high-throughput workflows.

Future Trends and Potential Uses

• Integration of real-time feedback control to adjust laser parameters based on precursor depletion.
• Combined UVPD with complementary dissociation methods (e.g., ETD, HCD) for multi-dimensional top-down analysis.
• Application to complex protein assemblies and high-mass proteoforms leveraging improved laser optics.
• Automation of fill/laser-pulse sequencing for standardized clinical and industrial workflows.

Conclusion

UVPD on the Orbitrap Fusion Tribrid system efficiently fragments intact proteins with high sequence coverage. The high-pressure cell is the preferred site for balanced fragmentation, and optimized multiple fill/laser-pulse strategies enable reproducible, high-quality top-down data. Proper laser energy control and ion protection are critical for robust performance.

Used Instrumentation

• Thermo Fisher Scientific Orbitrap Fusion Tribrid Mass Spectrometer
• 193 nm ArF excimer laser (Coherent ExciStar XS), energy feedback control, UV-transparent window flange

References

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3. Fellers RT et al. Proteomics. 2015;15(7):1235–1238.
4. Holden DD et al. Poster presented at ASMS Conference, Baltimore, MD, 2014.