Ultrahigh-Mass Resolution Mass Spectrometry Imaging with an Orbitrap Externally Coupled to a High-Performance Data Acquisition System.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool that enables molecular sample analysis while simultaneously providing the spatial context of hundreds or even thousands of analytes. However, because of the lack of a separation step prior to ionization and the immense diversity of biomolecules, such as lipids, including numerous isobaric species, the coupling of ultrahigh mass resolution (UHR) with MSI presents one way in which this complexity can be resolved at the spectrum level. Until now, UHR MSI platforms have been restricted to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Here, we demonstrate the capabilities of an Orbitrap-based UHR MSI platform to reach over 1,000,000 mass resolution in a lipid mass range (600-950 Da). Externally coupling the Orbitrap Q Exactive HF with the high-performance data acquisition system FTMS Booster X2 provided access to the unreduced data in the form of full-profile absorption-mode FT mass spectra. In addition, it allowed us to increase the time-domain transient length from 0.5 to 10 s, providing improvement in the mass resolution, signal-to-noise ratio, and mass accuracy. The resulting UHR performance generates high-quality MALDI MSI images and simplifies the identification of lipids. Collectively, these improvements resulted in a 1.5-fold increase in annotations, demonstrating the advantages of this UHR imaging platform for spatial lipidomics using MALDI-MSI.