Exploring Metabolic Pathways in Vivo by a Combined Approach of Mixed Stable Isotope-Labeled Raman Microspectroscopy and Multivariate Curve Resolution Analysis

Venkata Hemanth Nag Noothalapati, Shinsuke Shigeto

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Abstract

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.
Original languageEnglish
JournalAnalytical Chemistry
Volume86
Issue number15
DOIs
StatePublished - 5 Aug 2014

Keywords

  • NONNEGATIVE MATRIX FACTORIZATION; YEAST-CELL CYCLE; ETHANOL-PRODUCTION; FUNGAL BIOMASS; FLUX ANALYSIS; AMINO-ACIDS; SILAC; SPECTROSCOPY; ERGOSTEROL; MICROSCOPY

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