Implementation of 213 nm Ultra Violet Photodissociation (UVPD) on a Modified Orbitrap Fusion Lumos

Significance of the Topic

Ultraviolet photodissociation (UVPD) at 213 nm offers extensive sequence coverage and preservation of labile post‐translational modifications, making it a valuable tool for top‐down proteomics, biotherapeutic characterization, and advanced QA/QC laboratories.

• Generates a,b,c,x,y,z ion series for comprehensive backbone mapping.
• Maintains labile modifications such as phosphorylation and glycosylation.
• Meets growing demands for detailed proteome analysis and structural insights.

Objectives and Study Overview

This work details the integration of a compact 213 nm UVPD laser into a modified Thermo Scientific Orbitrap Fusion Lumos and evaluates its performance for top‐down sequencing of standard proteins.

• Implement a stable UVPD module requiring minimal user intervention.
• Assess alignment stability and laser performance metrics.
• Quantify sequence coverage across activation parameters and compare to ETD, CID, and HCD.

Methodology and Instrumentation

Standard proteins (apomyoglobin, carbonic anhydrase II) were prepared at 1 pmol/µL in 50/50 MeOH/H₂O with 0.1% formic acid and infused at 3 µL/min.

• Laser: CryLaS Q3 passively Q-switched Nd:YAG, 5th harmonic at 213 nm, output 3.75 ± 0.5 mW, pulse energy 1.5 ± 0.2 µJ at 2.5 kHz, <1 ns pulse width, beam ~450 µm.
• Integration: Mounted to dual‐cell linear ion trap via UV-grade fused silica optics and vacuum interface window.
• Acquisition: AGC 5e5, Orbitrap resolution 120 k, averaging 100 scans in low‐pressure mode (3 mTorr), variable UVPD irradiation times.
• Data Analysis: Deconvolution with Hardklör; ProSight Lite search at 10 ppm tolerance.

Used Instrumentation

• Thermo Scientific Orbitrap Fusion Lumos mass spectrometer with modified dual‐cell linear ion trap.
• CryLaS Q3 Nd:YAG laser delivering 213 nm UVPD.
• UV-grade fused silica steering optics and vacuum window assembly.

Main Results and Discussion

• Alignment Stability: Achieved consistent beam‐ion cloud overlap (AQS ≈ 0.5) with long‐term stability demonstrated over extended operation.
• Sequence Coverage: Optimized UVPD delivered ~55% coverage for apomyoglobin and ~30% for carbonic anhydrase II, matching or exceeding ETD, CID, and HCD under comparable conditions.
• Bond Coverage: UVPD provided unique fragmentation contributing to ~95% combined bond coverage when integrated with other methods.
• Performance: The module exhibited minimal maintenance requirements and stable pulse-to-pulse energy variation within ±0.2 µJ.

Benefits and Practical Applications

Integration of 213 nm UVPD enhances top-down workflows by improving sequence and PTM mapping, reducing instrument downtime, and supporting rigorous analysis in research, QA/QC, and biotherapeutic laboratories.

Future Trends and Applications

Prospective developments include broader adoption of UVPD on diverse platforms, advanced informatics for high‐throughput proteome studies, and innovations in laser design to further shrink system footprint and enhance stability.

Conclusion

The successful incorporation of a compact 213 nm UVPD module into an Orbitrap Fusion Lumos instrument demonstrates reliable performance, high sequence coverage, and robust operation, establishing UVPD as a powerful approach for top-down proteomics and biotherapeutic analysis.

References

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