Multiple Infusion Start Time Mass Spectrometry Imaging of Dynamic SIL-Glutathione Biosynthesis Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization

Abstract

Due to the high association of glutathione metabolism perturbation with a variety of disease states, there is a dire need for analytical techniques to study glutathione kinetics. Additionally, the elucidation of microenvironmental effects on changes in glutathione metabolism would significantly improve our understanding of the role of glutathione in disease. We therefore present a study combining a multiple infusion start time protocol, stable isotope labeling technology, infrared matrix-assisted laser desorption electrospray ionization, and high-resolution accurate mass−mass spectrometry imaging to study spatial changes in glutathione kinetics across in sectioned mouse liver tissues. After injecting a mouse with the isotopologues [2-13 C, 15 N]-glycine, [1,2-13 C 2 ]-glycine, and [1,2-13 C 2 , 15 N]-glycine at three different time points, we were able to fully resolve and spatially map their metabolism into three isotopologues of glutathione and calculate their isotopic enrichment in glutathione. We created a tool in the open-source mass spectrometry imaging software MSiReader to accurately compute the percent isotope enrichment (PIE) of these labels in glutathione and visualize them in heat-maps of the tissue sections. In areas of high flux, we found that each label enriched an approximate median of 1.6%, 1.8%, and 1.5%, respectively, of the glutathione product pool measured in each voxel. This method may be adapted to study the heterogeneity of glutathione flux in diseased versus healthy tissues.

On the importance of color in mass spectrometry imaging

“…The heatmap of glutathione (GSH, [M-H] À = m/z 306.0765) abundance normalized to the abundance of homoglutathione (hGSH, [M-H] À = m/z 320.0922) measured by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI)-MSI from a section of mouse liver was generated using the jet colormap and is shown in Figure 1A. 18 Homoglutathione was sprayed on the sample's microscope slide prior to sample mounting. The normalized GSH abundance is a relative quantification for GSH concentration within the tissue.…”

Highlighting Functional Mass Spectrometry Imaging Methods in Bioanalysis

“…To address this issue, we have adapted a timed substrate infusion method, first described by Dudley et al, and later coined Multiple Infusion Start Times (MIST) by Paolini et al, that has been used in mass spectrometry analysis to provide detailed labeling kinetic data from a single tissue sample. − Although not previously used with MSI, the MIST protocol is ideal for use in fMSI studies as a single tissue sample provides labeling data from multiple time points. Our study of mouse liver demonstrated that matrix-assisted laser desorption electrospray ionization (MALDESI) MSI can distinguish both the glycine isotopologue substrates and the glutathione products of metabolism . This method may likely be adapted to any system if multiple isotopologues of substrate molecules are available.…”

“…Our study of mouse liver demonstrated that matrix-assisted laser desorption electrospray ionization (MALDESI) MSI can distinguish both the glycine isotopologue substrates and the glutathione products of metabolism. 36 This method may likely be adapted to any system if multiple isotopologues of substrate molecules are available. This work was performed with an orbitrap instrument allowing for high resolving power measurements, a key to measuring separate isotopologues.…”

Sequential paired covariance for improved visualization of mass spectrometry imaging datasets

“…This can result in pixelated ion images making it difficult to delineate edges of histological features. To account for this, molecular standards with similar ionization efficiencies to the analyte of interest can be sprayed underneath biological samples and used for normalization 3–6 . However, normalization standards are difficult to choose for untargeted experiments because of the variability of ionization efficiencies between the molecules detected.…”

“…To account for this, molecular standards with similar ionization efficiencies to the analyte of interest can be sprayed underneath biological samples and used for normalization. 3 , 4 , 5 , 6 However, normalization standards are difficult to choose for untargeted experiments because of the variability of ionization efficiencies between the molecules detected. Many postprocessing normalization methods available do not require a standard to reduce the relative standard deviation between pixels/voxels.…”