IMPROVEMENTS IN A MULTI-REFLECTING TOF MASS SPECTROMETER TO ENHANCE MASS SPECTROMETRY IMAGING SPECIFICITY

Significance of the Topic

Mass spectrometry imaging (MSI) delivers spatially resolved chemical information but often encounters isobaric interferences when high spatial resolution is required. Increasing mass resolving power directly improves specificity, enabling more accurate molecular identification in complex biological tissues.

Objectives and Study Overview

This study presents a novel configuration of a multi-reflecting time-of-flight (MRT) mass spectrometer operated in a double-pass Resolution Enhancement Mode (REM) to achieve resolving power above 300,000 full width at half maximum (FWHM). The approach is demonstrated through lipid imaging in mouse kidney and brain sections.

Experimental Methodology

• Sample Preparation: Cryo-sectioning of healthy wild-type mouse kidney and brain at 16 µm thickness onto glass slides.
• Imaging Technique: Desorption electrospray ionization (DESI) using a DESI XS source.
• Acquisition Parameters: Positive ion mode, flow rate 2 µL/min (95:5 MeOH:water) with 100 pg/µL Leu-enkephalin, 0.86 kV spray voltage, 15 psi nebulizing gas, heated transfer line at 350 °C, two spectra per second.
• Data Analysis: Waters HDI v1.7 software for segmentation and putative identification based on LipidMaps database.

Instrumentation Used

• SELECT SERIES™ Multi-Reflecting TOF mass spectrometer, Waters Corporation.
• Orthogonal accelerator and multi-reflecting analyzer with lenses P1 and P23.
• Timed pulse synchronization between upstream gas cell and orthogonal accelerator to enable Resolution Enhancement Mode.

Key Results and Discussion

• REM doubled the effective ion pathlength to ~92 m compared to ~47 m in standard MRT, raising resolving power from ~200,000 to ~300,000 FWHM.
• Baseline separation of four lipid ions within a 30 mDa window was achieved only in REM mode, improving image clarity and identification confidence.
• In brain tissue, two species with a 2.8 mDa mass difference were distinguished when applying a 1 mDa mass window in REM data processing.
• Mass accuracy across selected lipids remained excellent, averaging 74 ppb RMS error.

Benefits and Practical Applications

• Enhanced mass specificity reduces spectral interferences, leading to more reliable molecular images.
• Improved lipidomics and biomarker discovery workflows in biological and clinical research.
• Maintains high throughput via synchronized ion packet timing without loss of duty cycle.

Future Trends and Potential Applications

Further developments may expand the accessible mass range, integrate alternative ionization sources, and implement adaptive REM parameters in real time. Applications could extend to pharmacokinetic mapping, pathology diagnostics, and single-cell MSI studies.

Conclusion

The double-pass REM configuration on a multi-reflecting TOF mass spectrometer substantially enhances MSI specificity by achieving over 300,000 FWHM resolving power and sub-ppm mass accuracy, unlocking detailed molecular insights in tissue imaging.

References

1. Cooper-Shepherd DA et al. Novel Hybrid Quadrupole-Multireflecting TOF Mass Spectrometry System. J Am Soc Mass Spectrom. 2023;34(2):264–272.