Simultaneous quantification of metabolites involved in central carbon and energy metabolism using reversed-phase liquid chromatography-mass spectrometry and in vitro 13C labeling

Wen Chu Yang, Miroslav Sedlak, Fred E. Regnier, Nathan Mosier, Nancy Ho, Jiri Adamec

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

Comprehensive analysis of intracellular metabolites is a critical component of elucidating cellular processes. Although the resolution and flexibility of reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) makes it one of the most powerful analytical tools for metabolite analysis, the structural diversity of even the simplest metabolome provides a formidable analytical challenge. Here we describe a robust RPLC-MS method for identification and quantification of a diverse group of metabolites ranging from sugars, phosphosugars, and carboxylic acids to phosphocarboxylics acids, nucleotides, and coenzymes. This method is based on in vitro derivatization with a 13C-labeled tag that allows internal standard based quantification and enables separation of structural isomer pairs like glucose 6-phosphate and fructose 6-phosphate in a single chromatographic run. Calibration curves for individual metabolites showed linearity ranging over more than 2 orders of magnitude with correlation coefficients of R2 > 0.9975. The detection limits at a signalto-noise ratio of 3 were below 1.0 μM (20 pmol) for most compounds. Thirty common metabolites involved in glycolysis, the pentose phosphate pathway, and tricarboxylic acid cycle were identified and quantified from yeast lysate with a relative standard deviation of less than 10%.

Original languageEnglish (US)
Pages (from-to)9508-9516
Number of pages9
JournalAnalytical chemistry
Volume80
Issue number24
DOIs
StatePublished - Dec 15 2008

ASJC Scopus subject areas

  • Analytical Chemistry

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