Notes & Tips In vivo analysis of intracellular amino acid labelings by GC/MS Christoph Wittmann, * Michael Hans, and Elmar Heinzle Biochemical Engineering Institute, Saarland University, P.O. Box 151150, 66041 Saarbruecken, Germany Received 27 December 2001 Detailed knowledge on carbon flux distributions is crucial for the understanding and targeted optimization of cellular systems. Analytical methods on quantification of fluxes through different pathways of a metabolic net- work are therefore in the core of metabolic engineering. An elegant approach for metabolic flux analysis is pro- vided by 13 C tracer experiments. As shown, amino acid labelings contain valuable information for the calcula- tion of metabolic fluxes [1–3]. The methods developed are mainly based on protein hydrolysates and therefore fail to resolve dynamics of metabolic fluxes, which are of great interest when profiling industrially relevant batch or fed-batch cultures, cell aging, or cell cycle phenomena. In contrast to proteins, free intracellular amino acids are permanently synthesized and consumed. In tracer stud- ies, their labeling patterns therefore reflect the actual metabolic state of a cell and provide information on metabolic fluxes under dynamic conditions. A few re- ports so far describe the labeling analysis of intracellular amino acids extracted from tissue, such as liver or heart, but include only a small number of compounds and ra- ther laborious protocols [4–6]. The rather low concen- trations of free intracellular amino acids in the millimolar range require sensitive methods for their analysis. In the present paper we combined a method recently developed for the extraction of free intracellular amino acids by Hans et al. [7] with t-butyldimethylsilyl (TBDMS) 1 de- rivatization and GC/MS as a novel tool for fast, accurate, and sensitive labeling analysis of free intracellular amino acids. TBDMS derivatization of amino acids is especially useful in metabolic network analysis, because [M-57] fragments containing the entire carbon skeleton of the analyte can be observed by GC/MS with high signal in- tensities [8]. The potential of the method is exemplified for Saccharomyces cerevisiae, certainly one of the most extensively studied microorganisms [9–11]. Materials and methods Organisms and growth conditions. S. cerevisiae ATCC 32167 was purchased from the American Type Strain and Culture Collection (Manassas, USA) and cultivated in continuous culture at a dilution rate of D ¼ 0:1h 1 in a 100-ml bioreactor (Meredos, Bovenden, Germany) at 30 °C and pH 6.0 on a synthetic medium as described previously [7], except for a decreased glucose concen- tration of 2 g/L. Sample preparation. Intracellular amino acids from S. cerevisiae were extracted by a specifically designed protocol that involved immediate quenching of meta- bolic activities, cell concentration by centrifugation, and subsequent extraction in boiling water [7]. For this purpose, cultivation samples containing 3.5mg biomass were injected into 50 ml of )40 °C cold buffered meth- anol. The obtained cell extract was purified from cell debris via centrifugation, lyophilized, and resuspended in 40 ll deionized water. Derivatization. Ten microliters of resuspended cell extract was lyophilized, subsequently dissolved in 8 ll dimethyl formamide containing 0.1% pyridine followed by addition of 8 ll N-methyl-N-t-butyldimethylsilyl- trifluoroacetamide (MBDSTFA) (Macherey-Nagel, D€ uren, Germany), and incubated for 60 min at 80 °C. The scheme of the derivatization reaction of an amino acid with MBDSTFA is shown in Fig. 1. GC/MS analysis. GC/MS measurements were carried out on a GC with an HP5MS capillary column (5% phenyl-methyl-siloxane diphenylpolysiloxane, 30m  250 lm), electron impact ionization at 70eV, and a quadrupole detector (Agilent Technologies, Waldbronn, Germany). The optimized conditions for the measure- ment of amino acids in cell extracts were as follows. The injected sample volume was 0.2 ll. Carrier gas flow was helium at 1.5 ml min 1 . The temperature gradient for the Analytical Biochemistry 307 (2002) 379–382 www.academicpress.com ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: +49-681-302-4572. E-mail address: c.wittmann@mx.uni-saarland.de (C. Wittmann). 1 Abbreviations used: TBDMS, t-butyldimethylsilyl; MBDSTFA, N-methyl-N-t-butyldimethylsilyl-trifluoroacetamide. 0003-2697/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0003-2697(02)00030-1