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Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of [13C6]nicotinamide coupled to MALDI-MSI (Iso-imaging) in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using [13C6]glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo.

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.

Plasma metabolite concentrations and labeling enrichments are common measures of organismal metabolism. In mice, blood is often collected by tail snip sampling. Here, we systematically examined the effect of such sampling, relative to gold-standard sampling from an in-dwelling arterial catheter, on plasma metabolomics and stable isotope tracing. We find marked differences between the arterial and tail circulating metabolome, which arise from two major factors: handling stress and sampling site, whose effects were deconvoluted by taking a second arterial sample immediately after tail snip. Pyruvate and lactate were the most stress-sensitive plasma metabolites, rising ~14 and ~5-fold. Both acute handling stress and adrenergic agonists induce extensive, immediate production of lactate, and modest production of many other circulating metabolites, and we provide a reference set of mouse circulatory turnover fluxes with noninvasive arterial sampling to avoid such artifacts. Even in the absence of stress, lactate remains the highest flux circulating metabolite on a molar basis, and most glucose flux into the TCA cycle in fasted mice flows through circulating lactate. Thus, lactate is both a central player in unstressed mammalian metabolism and strongly produced in response to acute stress. [ABSTRACT FROM AUTHOR]

Cellular heterogeneity is an inherent property of cell populations with a wide spectrum of biological manifestations, ranging from barely observable variations that enhance organismal adaptation, to life‐threatening differences. Single‐cell metabolomics can reveal molecular information and variations in metabolite concentrations between cells that are masked in cell‐population studies. These differences are quantitatively captured by the abundance distributions for the population and their statistical analysis can reveal the presence of latent subpopulations. In recent years, mass spectrometry (MS), combined with novel sampling and ionization techniques, has become an important tool for single‐cell metabolomics. These new techniques must contend with significant challenges in the form of small cell sizes and volumes, ultratrace metabolite amounts per cell, and potentially interfering high turnover rates and rapid diffusion. Owing to the ultrasmall sample volume, low abundance of some metabolites, and poor ionization efficiencies, metabolite detection, identification, and accurate quantitation remain a major challenge. The ability of some techniques to analyze tissue‐embedded cells opens the door for spatial metabolomics, potentially revealing cellular synergism in organ level metabolism. In this Concept article, we present a bird's eye view of these major themes in single cell metabolomics and some of the relevant MS‐based methods. [ABSTRACT FROM AUTHOR]

Mass spectrometry imaging (MSI) plays an expanding role in the label-free spatial mapping of hundreds of molecules simultaneously. Currently, matrix-assisted laser desorption ionization (MALDI) is among the most widely adopted MSI techniques. However, matrix application can impact the fidelity of spatial distributions, and matrix selection and related spectral interferences in the low mass range can lead to biased molecular coverage. Nanophotonic ionization from silicon nanopost arrays (NAPA) is an emerging matrix-free MSI platform with enhanced sensitivity for several molecular classes, for example, neutral lipids and biooligomers. Here, we describe a protocol with minimal sample preparation for NAPA-MSI of metabolites, lipids, and biooligomers from biological tissues., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Metabolomic measurements can provide functional readouts of cellular states and phenotypes. Here, we present a protocol for single-cell metabolomics that permits direct untargeted detection of a broad number of metabolites under ambient conditions, without the need for sample processing, and with high confidence in the discovery and identification of the molecular formulas for detected metabolites. This protocol describes combining fiber-based laser ablation electrospray ionization (f-LAESI) with a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer (21T-FTICR-MS) to obtain high confidence molecular formula information about detected metabolites. The f-LAESI source utilizes mid-infrared laser ablation through a sharp optical fiber tip, affording direct ambient analysis of cells without the need for sample processing. Using the 21T-FTICR-MS as a mass analyzer enabled measurement of the isotopic fine structure (IFS) for numerous metabolites simultaneously from single cells, and the IFSs were in turn computationally processed to rapidly determine the corresponding elemental compositions. This metabolomics technique complements other single cell omics measurement methods, helping to resolve complex molecular interactions that take place within cells unattainable from single cell transcriptomic and proteomics methods., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)