Non-invasively probing metabolites within single live cells is highly desired but challenging. Here we utilize Raman spectro-microscopy for spatial mapping of metabolites within single cells, with the specific goal of identifying druggable metabolic susceptibilities from a series of patient-derived melanoma cell lines. Each cell line represents a different characteristic level of cancer cell de-differentiation. First, with Raman spectroscopy, followed by stimulated Raman scattering (SRS) microscopy and transcriptomics analysis, we identify the fatty acid synthesis pathway as a druggable susceptibility for differentiated melanocytic cells. We then utilize hyperspectral-SRS imaging of intracellular lipid droplets to identify a previously unknown susceptibility of lipid mono-unsaturation within de-differentiated mesenchymal cells with innate resistance to BRAF inhibition. Drugging this target leads to cellular apoptosis accompanied by the formation of phase-separated intracellular membrane domains. The integration of subcellular Raman spectro-microscopy with lipidomics and transcriptomics suggests possible lipid regulatory mechanisms underlying this pharmacological treatment. Our method should provide a general approach in spatially-resolved single cell metabolomics studies.

SignificanceThe deuterium/protium ( 2 H/ 1 H) ratio of microbial lipids varies substantially and appears to be correlated with the mode of metabolism in the host organism, with lipids from chemoautotrophs and photoautotrophs 2 H-depleted and heterotrophs in many cases 2 H-enriched. Such patterns suggest that the Hisotope ratios of lipids could be used to infer the metabolism of environmental organisms, with applications ranging from Earth history to global carbon cycling and ecology. Here, we learn to understand this information by using metabolic flux analysis. We show that the full range of lipid 2 H/ 1 H ratios from a diverse set of aerobic heterotrophs can be quantitatively explained by fluxes through various NADP + -reducing reactions due to differences in their kinetic isotope effects.

Assessing the fate of nitroaromatic explosives in the subsurface is challenging because contaminants are present in different phases (e.g., bound to soil or sediment matrix or as solid-phase residues) and transformation takes place via several potentially competing pathways over time-scales of decades. We developed a procedure for compound-specific analysis of stable C, N, and H isotopes in nitroaromatic compounds (NACs) and characterized biodegradation of 2,4,6-trinitrotoluene (TNT) and two dinitrotoluene isomers (2,4-DNT and 2,6-DNT) in subsurface material of a contaminated site. The type and relative contribution of reductive and oxidative pathways to the degradation of the three contaminants was inferred from the combined evaluation of C, N, and H isotope fractionation. Indicative trends of Δδ(15)N vs Δδ(13)C and Δδ(2)H vs Δδ(13)C were obtained from laboratory model systems for biodegradation pathways initiated via (i) dioxygenation, (ii) reduction, and (iii) CH3-group oxidation. The combined evaluation of NAC isotope fractionation in subsurface materials and in laboratory experiments suggests that in the field, 86-89% of 2,4-DNT transformation was due to dioxygenation while TNT was mostly reduced and 2,6-DNT reacted via a combination of reduction and CH3-group oxidation. Based on historic information on site operation, our data imply biodegradation of 2,4-DNT with half-lives of up to 9-17 years compared to 18-34 years for cometabolic transformation of TNT and 2,6-DNT.

Oxidation of aromatic rings and its alkyl substituents are often competing initial steps of organic pollutant transformation. The use of compound-specific isotope analysis (CSIA) to distinguish between these two pathways quantitatively, however, can be hampered by large H isotope fractionation that precludes calculation of apparent (2)H-kinetic isotope effects (KIE) as well as the process identification in multi-element isotope fractionation analysis. Here, we investigated the C and H isotope fractionation associated with the transformation of toluene, nitrobenzene, and four substituted nitrotoluenes by permanganate, MnO4(-), to propose a refined evaluation procedure for the quantitative distinction of CH3-group oxidation and dioxygenation. On the basis of batch experiments, an isotopomer-specific kinetic model, and density functional theory (DFT) calculations, we successfully derived the large apparent (2)H-KIE of 4.033 ± 0.20 for the CH3-group oxidation of toluene from H isotope fractionation exceeding >1300‰ as well as the corresponding (13)C-KIE (1.0324 ± 0.0011). Experiment and theory also agreed well for the dioxygenation of nitrobenzene, which was associated with (2)H- and (13)C-KIEs of 0.9410 ± 0.0030 (0.9228 obtained by DFT) and 1.0289 ± 0.0003 (1.025). Consistent branching ratios for the competing CH3-group oxidation and dioxygenation of nitrotoluenes by MnO4(-) were obtained from the combined modeling of concentration as well as C and H isotope signature trends. Our approach offers improved estimates for the identification of contaminant microbial and abiotic oxidation pathways by CSIA.