Understanding cellular metabolism is a major challenge in current systems biology and has triggered extensive metabolomics research, which in most cases involves destructive analysis. However, the information obtainable only in a nondestructive manner will be required for accurately mapping the global structure of the organism's metabolic network at a given instant. Here we report that metabolic pathways can be explored in vivo by mixed stable isotope-labeled Raman microspectroscopy in conjunction with multivariate curve resolution analysis. As a model system, we studied ergosterol biosynthesis in single living fission yeast cells grown in mixtures of normal and C-13-labeled glucose as the sole carbon source. The multivariate spectral data analysis of space-resolved Raman spectra revealed the intrinsic spectra and relative abundances of all isotopomers of ergosterol whose carbon atoms in the 5,7-diene moiety of the sterol skeleton are either partly or fully substituted with C-13. Our approach is applicable to other metabolites and will earn a place in the toolbox of metabolomic analysis.
- NONNEGATIVE MATRIX FACTORIZATION; YEAST-CELL CYCLE; ETHANOL-PRODUCTION; FUNGAL BIOMASS; FLUX ANALYSIS; AMINO-ACIDS; SILAC; SPECTROSCOPY; ERGOSTEROL; MICROSCOPY
Hemanth Nag Noothalapati, V., & Shigeto, S. (2014). Exploring Metabolic Pathways in Vivo by a Combined Approach of Mixed Stable Isotope-Labeled Raman Microspectroscopy and Multivariate Curve Resolution Analysis. Analytical Chemistry, 86(15). https://doi.org/10.1021/ac501735c