Metal-Assisted Laser Desorption Ionization Imaging Mass Spectrometry.

Matrix-assisted laser desorption ionization (MALDI) remains the reference method to generate molecular images of proteins and lipids within thin tissue sections. However, traditional MALDI imaging mass spectrometry (IMS) suffers from low matrix homogeneity and high signal background in low mass range caused by matrix signals. To overcome these issues, alternative workflow and methods have been developed. Of these, metal-assisted laser desorption ionization (LDI) has become a reference technique to ionize low molecular weight compounds while allowing IMS at very high spatial resolutions with very low background signal in the low mass range. Silver and gold remain the two most used metals for the detection of neutral lipids including cholesterol, free fatty acids, and triglycerides. In this chapter, we demonstrate the potential of metal-assisted LDI IMS through the analysis of spinal cord and kidney thin tissue sections after silver and gold metal deposition. We also detail typical step-by-step workflows and discuss the strength of the methods.

Minimizing Visceral Fat Delocalization on Tissue Sections with Porous Aluminum Oxide Slides for Imaging Mass Spectrometry.

A high correlation of bioanalytes with their corresponding histologies is the landmark feature of matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). Lipids are one of the most studied classes of biomolecules, and monitoring lipid distribution and abundance in tissue samples can lead to major inputs in the understanding of disease. Lipid delocalization and ion suppression are two major effects that can lead to misinterpretation of the IMS results to an unaware analyst. We and others have observed that tissue specimens containing high amounts of visceral fat are challenging to analyze because of fat delocalization on and off section leading to significant triacylglyceride and phospholipid delocalization and major ion suppression effects. In this work, we introduce a novel and easy to produce reusable porous aluminum oxide sample slide that minimizes visceral fat delocalization after thaw-mounting of tissue sections. Using fatty mouse kidneys and other tissues, we demonstrate its efficacy in minimizing delocalization of triacylglycerides, the primary constituents of fat, and the resulting beneficial effects on phospholipid MALDI IMS.

Silver spray deposition for AgLDI imaging MS of cholesterol and other olefins on thin tissue sections.

Olefins such as cholesterol and unsaturated fatty acids play important biological roles. Silver-assisted laser desorption ionization (AgLDI) takes advantage of the strong affinity of silver to conjugate with double bonds to selectively ionize these molecules for imaging mass spectrometry (IMS) experiments. For IMS studies, two main approaches for silver deposition have been described in the literature: fine coating by silver sputtering and spray deposition of silver nanoparticles. While these approaches allow for extremely high resolution IMS experiments to be conducted, they are not readily available to all laboratories. Herein, we present a silver nitrate spray deposition approach as an alternative to silver sputtering and nanoparticle deposition for routine IMS analysis. The silver nitrate spray has the same level of specificity and sensitivity for olefins, particularly cholesterol, and has shown to be capable of IMS experiments down to 10-µm spatial resolution. Minimal sample preparation and the affordability of silver nitrate make this a convenient and accessible technique worth considering.

Mild Methylenetetrahydrofolate Reductase Deficiency Alters Inflammatory and Lipid Pathways in Liver.

SCOPE: Dietary and genetic folate disturbances can lead to nonalcoholic fatty liver disease (NAFLD). A common variant in methylenetetrahydrofolate reductase (MTHFR 677C→T) causes mild MTHFR deficiency with lower 5-methyltetrahydrofolate for methylation reactions. The goal is to determine whether mild murine MTHFR deficiency contributes to NAFLD-related effects. METHODS AND RESULTS: Wild-type and Mthfr+/- mice, a model for the human variant, are fed control (CD) or high-fat (HFAT) diets for 8 weeks. On both diets, MTHFR deficiency results in decreased S-adenosylmethionine, increased S-adenosylhomocysteine, and decreased betaine with reduced methylation capacity, and changes in expression of several inflammatory or anti-inflammatory mediators (Saa1, Apoa1, and Pon1). On CD, MTHFR deficiency leads to microvesicular steatosis with expression changes in lipid regulators Xbp1s and Cyp7a1. The combination of MTHFR deficiency and HFAT exacerbates changes in inflammatory mediators and introduces additional effects on inflammation (Saa2) and lipid metabolism (Nr1h4, Srebf1c, Ppara, and Crot). These effects are consistent with increased expression of pro-inflammatory HDL precursors and greater lipid accumulation. MTHFR deficiency may enhance liver injury through alterations in methylation capacity, inflammatory response, and lipid metabolism. CONCLUSION: Individuals with the MTHFR variant may be at increased risk for liver disease and related complications, particularly when consuming high-fat diets.