In mass spectrometry imaging (MSI) applications of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), an exogenous ice layer is the gold standard for an energy-absorbing matrix. However, the formation of the ice matrix requires additional time and instrument hardware, so glycerol was investigated herein as an alternative to the ice matrix to potentially improve spatial resolution and ionization, while decreasing experiment time. Glycerol solutions of varying concentrations were sprayed over top of rat liver tissue sections for analysis by IR-MALDESI and compared to the typical ice matrix condition. Additionally, we tested if combining the ice matrix and glycerol matrix would further improve analyses. Matrix conditions were evaluated by comparing ion abundance of six lipid species, the laser ablation spot diameter, and number of METASPACE annotations. The ion abundances were also normalized to the volume of tissue ablated to correct for lower abundance values due to less ablated tissue. It was observed that utilizing a 50% glycerol matrix without ice provides improved spatial resolution with lipid abundances and annotations comparable to the ice matrix standard, while decreasing the time required to complete an IR-MALDESI tissue imaging experiment.

Most mass spectrometry imaging (MSI) methods provide a molecular map of tissue content but little information on tissue function. Mapping tissue function is possible using several well-known examples of “functional imaging” such as positron emission tomography and functional magnetic resonance imaging that can provide the spatial distribution of time-dependent biological processes. These functional imaging methods represent the net output of molecular networks influenced by local tissue environments that are difficult to predict from molecular/cellular content alone. However, for decades, MSI methods have also been demonstrated to provide functional imaging data on a variety of biological processes. In fact, MSI exceeds some of the classic functional imaging methods, demonstrating the ability to provide functional data from the nanoscale (subcellular) to whole tissue or organ level. This Perspective highlights several examples of how different MSI ionization and detection technologies can provide unprecedented detailed spatial maps of time-dependent biological processes, namely, nucleic acid synthesis, lipid metabolism, bioenergetics, and protein metabolism. By classifying various MSI methods under the umbrella of “functional MSI”, we hope to draw attention to both the unique capabilities and accessibility with the aim of expanding this underappreciated field to include new approaches and applications.

By employing chip-based capillary zone electrophoresis coupled to high-resolution mass spectrometry, we profiled the plasma metabolome of 134 patients diagnosed with sporadic amyotrophic lateral sclerosis (ALS) (81 males and 53 females) and 118 individuals deemed healthy (49 males and 69 females). The most significant markers (p < 0.01) were creatine, which was 49% elevated, and creatinine and methylhistidine, which were decreased by 20 and 24%, respectively, in ALS patients. The ratio of creatine versus creatinine increased 370 and 200% for male and female ALS patients, respectively. In addition, male ALS patients on an average had 5–13% lower amounts of seven essential amino acids, whereas females did not significantly differ from healthy controls. We developed two models using the metabolite abundances: (1) a classification model for the separation of ALS and healthy samples and (2) a classification model for the prediction of disease progression based on the ALS functional rating score. Utilizing a Monte Carlo cross-validation approach, a linear discriminant analysis model achieved a mean area under the receiver operating characteristic curve (AUC) of 0.85 (0.06) with a mean sensitivity of 80% (9%) and specificity of 78% (10%) for the separation of ALS and controls, respectively. A support vector machine classifier predicted progression categories with an AUC of 0.90 (0.06) with a mean sensitivity of 73% (10%) and a specificity of 86% (5%). Lastly, using a previously reported assay with a stable isotope-labeled (13C3 15N2) spike-in standard, we were unable to detect the exogenous neurotoxic metabolite, β-methylamino-l-alanine, in the free or protein-bound fraction of any of the 252 plasma samples.

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.

In an effort to develop an analytical method capable of finding new metalloproteins, this is the first report of a new diagonal gel electrophoresis method to isolate and identify metalloproteins, based on the molecular recognition of holo- and apo-metalloproteins (metalbound and -free forms, respectively) by CBB G-250 dye and employing metal ion contaminant sweeping-blue native-polyacrylamide gel electrophoresis (MICS-BN-PAGE). The difference in electrophoretic mobilities between holo- and apo-forms was exaggerated as a result of interactions between the metalloproteins and the dye with no metal ion dissociation. The different binding modes of proteins with CBB G-250 dye, primarily related to hydrogen bonding, were confirmed by capillary zone electrophoresis (CZE) and molecular docking simulations. Due to in-gel holo/apo conversion between the first and second dimensions of PAGE, holo-metalloproteins in the original sample were completely isolated as spots off the diagonal line in the second dimension of PAGE. To prove the high efficiency of this method for metalloprotein analysis, we successfully identified a copper-binding protein from a total bacterial soluble extract for the first time.

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