期刊
ANALYTICAL CHEMISTRY
卷 80, 期 24, 页码 9508-9516出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac801693c
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资金
- U.S. Department of Energy Biomass Program [GO17059-16649]
- National Science Foundation [DBI-0421102]
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 C-13-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 R-2 > 0.9975. The detection limits at a signal-to-noise ratio of 3 were below 1.0 mu M (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%.
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