Most clinical studies of Cannabis today focus on the contents of two phytocannabinoids: (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), regardless of the fact that the plant contains over 100 additional phytocannabinoids whose therapeutic effects and interplay have not yet been fully elucidated. This narrow view of a complex Cannabis plant is insufficient to comprehend the medicinal and pharmacological effects of the whole plant. In this study we suggest a new ESI-LC/MS/MS approach to identify phytocannabinoids from 10 different subclasses, and comprehensively profile the identified compounds in diverse medical Cannabis plants. Overall, 94 phytocannabinoids were identified and used for profiling 36 of the most commonly used Cannabis plants prescribed to patients in Israel. In order to demonstrate the importance of comprehensive phytocannabinoid analysis before and throughout medical Cannabis clinical trials, treatments, or experiments, we evaluated the anticonvulsant effects of several equally high-CBD Cannabis extracts (50% w/w). We found that despite the similarity in CBD contents, not all Cannabis extracts produced the same effects. This study’s approach for phytocannabinoid profiling can enable researchers and physicians to analyze the effects of specific Cannabis compositions and is therefore critical when performing biological, medical and pharmacological-based research using Cannabis.

Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal-fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse-chase LC-MS-based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine-derived fluxes: Oxidation of glucosederived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation.

Mass spectrometry based metabolomics is a widely used approach in biomedical research. However, current methods coupling mass spectrometry with chromatography are timeconsuming and not suitable for high-throughput analysis of thousands of samples. An alternative approach is flow-injection mass spectrometry (FI-MS) in which samples are directly injected to the ionization source. Here, we show that the sensitivity of Orbitrap FI-MS metabolomics methods is limited by ion competition effect. We describe an approach for overcoming this effect by analyzing the distribution of ion m/z values and computationally determining a series of optimal scan ranges. This enables reproducible detection of~9,000 and~10,000 m/z features in metabolomics and lipidomics analysis of serum samples, respectively, with a sample scan time of~15 s and duty time of~30 s; a~50% increase versus current spectral-stitching FI-MS. This approach facilitates high-throughput metabolomics for a variety of applications, including biomarker discovery and functional genomics screens.

Most studies on the skin focus primarily on the hair follicle and interfollicular epidermis, whereas little is known regarding the homeostasis of the sebaceous gland (SG). The SG has been proposed to be replenished by different pools of hair follicle stem cells and cells that resides in the SG base, marked by Blimp1. Here, we demonstrate that single Blimp1 + cells isolated from mice have the potential to generate SG organoids in vitro. Mimicking SG homeostasis, the outer layer of these organoids is composed of proliferating cells that migrate inward, undergo terminal differentiation and generating lipid-filled sebocytes. Performing confocal microscopy and mass-spectrometry, we report that these organoids exhibit known markers and a lipidomic profile similar to SGs in vivo. Furthermore, we identify a role for c-Myc in sebocyte proliferation and differentiation, and determine that SG organoids can serve as a platform for studying initial stages of acne vulgaris, making this a useful platform to identify potential therapeutic targets.

The inability to inspect metabolic activities within subcellular compartments has been a major barrier to our understanding of eukaryotic cell metabolism. Here, we describe a spatial-fluxomics approach for inferring metabolic fluxes in mitochondria and cytosol under physiological conditions, combining isotope tracing, rapid subcellular fractionation, LC-MS-based metabolomics, computational deconvolution, and metabolic network modeling. Applied to study reductive glutamine metabolism in cancer cells, shown to mediate fatty acid biosynthesis under hypoxia and defective mitochondria, we find a previously unappreciated role of reductive IDH1 as the sole net contributor of carbons to fatty acid biosynthesis under standard normoxic conditions in HeLa cells. In murine cells with defective SDH, we find that reductive biosynthesis of citrate in mitochondria is followed by a reversed CS activity, suggesting a new route for supporting pyrimidine biosynthesis. We expect this spatial-fluxomics approach to be a highly useful tool for elucidating the role of metabolic dysfunction in human disease.