Experimental Assessment of Mammalian Lipidome Complexity Using Multimodal 21 T FTICR Mass Spectrometry Imaging

Abstract

Herein, we assess the complementarity and complexity of data that can be detected within mammalian lipidome mass spectrometry imaging (MSI) via matrix-assisted laser desorption ionization (MALDI) and nanospray desorption electrospray ionization (nano-DESI). We do so by employing 21 T Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with absorption mode FT processing in both cases, allowing unmatched mass resolving power per unit time (≥613k at m/z 760, 1.536 s transients). While our results demonstrated that molecular coverage and dynamic range capabilities were greater in MALDI analysis, nano-DESI provided superior mass error, and all annotations for both modes had sub-ppm error. Taken together, these experiments highlight the coverage of 1676 lipids and serve as a functional guide for expected lipidome complexity within nano-DESI-MSI and MALDI-MSI. To further assess the lipidome complexity, mass splits (i.e., the difference in mass between neighboring peaks) within single pixels were collated across all pixels from each respective MSI experiment. The spatial localization of these mass splits was powerful in informing whether the observed mass splits were biological or artificial (e.g., matrix related). Mass splits down to 2.4 mDa were observed (i.e., sodium adduct ambiguity) in each experiment, and both modalities highlighted comparable degrees of lipidome complexity. Further, we highlight the persistence of certain mass splits (e.g., 8.9 mDa; double bond ambiguity) independent of ionization biases. We also evaluate the need for ultrahigh mass resolving power for mass splits ≤4.6 mDa (potassium adduct ambiguity) at m/z > 1000, which may only be resolved by advanced FTICR-MS instrumentation.

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

“…The data acquisition and processing enhancements made to the MALDI QE HF MSI platform significantly improved the quality of MALDI imaging in this work. The maximum mass resolution attained at m / z 800 is ∼1,400,000, representing the highest resolution achieved to date for any Orbitrap-based imaging platform and at the level of work done with FT-ICR-based platforms. ,− An increase of 70% in the S/N ratio has been observed for lipid peaks with transient lengths of ∼2 s, which proved to be the optimal length for enhancing instrument sensitivity. Furthermore, the mass accuracy was also improved by an order of magnitude.…”

“…The attained mass resolution aligns with the research on FT-ICR-based UHR MSI platforms. Nevertheless, this is the first Orbitrap-based imaging platform that achieved such mass resolution for the MSI of lipids. ,,, …”

“…The chemical complexity of biological tissue samples can present a challenge for MSI as many signals may remain unresolved. For example, isobaric lipid ions, such as [13C2]­[PC 34:0 + Na] + and [PC 34:1 + Na] + , may lead to inaccurate identification, quantification, and spatial mapping. , Ultrahigh mass resolution (UHR) platforms, defined here as a mass resolution of over 500,000 at m / z 200 or exceeding 250,000 at m / z 800, may be able to resolve these isobaric signals. Orbitrap and ion cyclotron resonance (ICR) Fourier-transform (FT) mass spectrometers have demonstrated UHR performance. − Recent advances in multireflectron time-of-flight instrumentation have also enhanced their mass resolving power and brought them to the UHR level (>200,000 in the lipid mass range). , In addition to mass resolution, the ion mobility has also proven to be a powerful approach for resolving isobaric and some isomeric analytes, while recent developments in alternative ion activation techniques also enable the resolution of isomeric lipids that cannot be resolved with mass resolution alone. , …”

“…Recently, nanospray desorption electrospray ionization (nano-DESI), desorption electrospray ionization (DESI), laser ablation electrospray ionization (LAESI), and MALDI ion sources have been coupled with FT-ICR mass spectrometers and were shown to achieve mass resolution of 1,000,000 or more in the lipid mass range (600–950 Da). ,− However, FT-ICR MS instruments, especially those with high magnetic field strengths, such as 18–21 T, are uncommon.…”

Effects of Sex and Western Diet on Spatial Lipidomic Profiles for the Hippocampus, Cortex, and Corpus Callosum in Mice Using MALDI MSI

“…While these studies show the importance of lipids in neurological function, demonstrating clear changes in the overall lipidomic profile as a function of diet, the methods used require homogenization of the sample and prevent spatial analysis. Spatial mapping of lipidomic changes remains critical to understanding disease onset and progression since brain regions are differentially affected in many neurological disorders. − Studying lipid profiles using in situ mass spectrometry-based techniques are some of the most powerful methods to characterize lipid metabolism in tissues. − There are several types of mass spectrometry imaging (MSI) instruments enabling spatial lipidomic analysis, including secondary ion mass spectrometry (SIMS), nanospray desorption electrospray ionization (nano-DESI) MSI and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI) MSI. , MALDI MSI has grown in popularity for neuroscience applications because it provides direct metabolomic information within cells/brain regions in situ and can measure thousands of lipids, proteins, peptides, and more while maintaining high mass accuracy and spatial resolution. − MALDI MSI uses a UV laser to ionize and desorb molecules from the tissue surface while maintaining spatial distribution by rastering the laser across the tissue, resulting in ion images with a spatial resolution of ∼10 μm. Depending on tissue sample size, > 500,000 of multiplexed spectra containing hundreds or even thousands of ion species are collected, allowing for visualization of each ion’s relative abundance across the sample.…”