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Separation and Quantitation of Water Soluble Cellular Metabolites by Hydrophilic Interaction Chromatography-Tandem Mass Spectrometry Sunil U

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Separation and Quantitation of Water Soluble Cellular Metabolites by Hydrophilic Interaction Chromatography-Tandem Mass Spectrometry Sunil U Journal of Chromatography A, 1125 (2006) 76–88 Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry Sunil U. Bajad a, Wenyun Lu a, Elizabeth H. Kimball a, Jie Yuan a, Celeste Peterson b, Joshua D. Rabinowitz a,∗ a Lewis-Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University, Princeton, NJ 08544, USA b Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA Received 13 January 2006; received in revised form 8 May 2006; accepted 10 May 2006 Available online 6 June 2006 Abstract A key unmet need in metabolomics is the ability to efficiently quantify a large number of known cellular metabolites. Here we present a liquid chromatography (LC)–electrospray ionization tandem mass spectrometry (ESI-MS/MS) method for reliable measurement of 141 metabolites, including components of central carbon, amino acid, and nucleotide metabolism. The selected LC approach, hydrophilic interaction chromatography with an amino column, effectively separates highly water soluble metabolites that fail to retain using standard reversed-phase chromatography. MS/MS detection is achieved by scanning through numerous selected reaction monitoring events on a triple quadrupole instrument. When applied to extracts of Escherichia coli grown in [12C]- versus [13C]glucose, the method reveals appropriate 12C- and 13C-peaks for 79 different metabolites. © 2006 Elsevier B.V. All rights reserved. Keywords: LC–ESI-MS/MS; Escherichia coli; Metabolomics; Metabonomics; Hydrophilic interaction chromatography; Bacteria; Metabolite; metabolism; Triple quadrupole; Mass spectrometry 1. Introduction pounds, likely capture most key metabolic end products and intermediates [5–8]. Thus, a key current need is an assay that The ability to quantify numerous mRNA in parallel using can reliably and efficiently measure these known compounds DNA microarrays has revolutionized biological science, with [9]. Such measurement, however, is difficult, especially due to important applications ranging from grouping genes into func- the low abundance of most cellular metabolites, which in total tional pathways to predicting the progression of human dis- comprise only ∼3% of E. coli dry weight [10]. eases [1]. In the wake of the genomics revolution, there has Various approaches have been applied recently in an effort been a drive to bring similarly comprehensive analysis to other to measure multiple metabolites. These include thin-layer chro- aspects of biology, including metabolism [2–4]. Experimental matography [11,12], high-performance liquid chromatography metabolomics, however, suffers from its lack of a microarray (LC) with detection based on absorption or emission of light equivalent: a highly parallel assay for quantifying known cellu- [13], nuclear magnetic resonance spectroscopy [14–16] and lar metabolites. chromatography coupled to mass spectrometry (MS) [17–22]. A first challenge in attempting to quantify every component Among these approaches, chromatography coupled to MS of the metabolome of an organism is the absence of a master stands out for its potential for high sensitivity and specificity. template, analogous to the genome, for cellular small molecules. In addition, MS offers the opportunity to confirm the molecu- Nevertheless, analysis of the full genomes of unicellular model lar formulas of the specific compounds being measured and to organisms suggests that current metabolic maps, which include conduct isotope-ratio-based quantitation of biological samples for Escherichia coli some 500 different water soluble com- [23]. Chromatography-MS methods can be grouped according to the type of chromatography and MS performed. While gas ∗ Corresponding author. Tel.: +1 609 258 8985; fax: +1 609 258 3565. chromatography-MS has proven a powerful tool for evaluating E-mail address: [email protected] (J.D. Rabinowitz). numerous low molecular weight metabolites [22], it is not well 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.05.019 S.U. Bajad et al. / J. Chromatogr. A 1125 (2006) 76–88 77 suited to analyzing metabolites with low volatility and thermal Table 1 stability, such as phosphate-containing compounds. Thus, much Metabolites investigated current research focuses on LC–MS with electrospray ioniza- Amino acids tion. While straightforward LC–MS provides a useful tool for Alanine initial analysis of samples without prior knowledge of the ana- Arginine lytes of interest, for quantifying compounds with known frag- Asparagine Aspartate mentation patterns, selected reaction monitoring (SRM) using Cysteine a triple quadrupole instrument offers additional sensitivity and Glutamate specificity [24]. SRM involves selecting for ions of a specified Glutamine parent molecular weight (m/z ratio), fragmenting the parent ion Glycine at an optimal collision energy for producing a particular daughter Histidine (Iso)leucine ion, and then quantifying the production of ions of the daughter Lysine mass. By scanning through multiple SRM events, it is possible Methionine to measure numerous compounds in a single LC run with this Phenylalanine approach. Proline To date, major barriers to employing triple quadrupole Serine Threonine LC–MS/MS to quantify cellular metabolites included lack of Tryptophan knowledge of the SRM transitions of most metabolites and the Tyrosine absence of good LC methods for metabolite separation. Here, we Valine address both of these deficiencies for 141 metabolites (Table 1 Amino acid derivatives/precursors ), corresponding to ∼25% of the known metabolome of E. coli. 3-Phospho-serine These 141 compounds were selected based on their stability, Citrulline importance to core metabolic processes common also to eukary- Cystathionine Histidinol otic organisms, and the availability of purified standards of the Homocysteic acid compounds. They include, for example, 19 of the 20 proteogenic Homocysteine amino acids, 29 nucleotides, 10 components of glycolysis and Homoserine the tricarboxylic acid (TCA) cycle, and nearly every compound Hydroxyphenylpyruvate ␣ involved in de novo pyrimidine biosynthesis. Of these 141 com- N- -Acetylornithine Ornithine pounds, we are able to quantify 69 from E. coli extracts, and find p-Aminobenzoate/anthranilate significant changes in 39 upon carbon starvation of E. coli. Phenylpyruvate Prephenate 2. Experimental S-Adenosyl-l-homocysteine S-Adenosyl-l-methionine Shikimate 2.1. Chemicals, reagents, and chromatography columns Taurine Nucleoside bases HPLC-grade solvents (OmniSolv, EMD Chemical, Gibb- Adenine stown, NJ, USA) were obtained from VWR International (West Cytosine Chester, PA, USA); ammonium acetate (99.4%) and formic acid Guanine (88%) from Mallinckrodt Chemicals, Phillipsburg, NJ, USA; Hypoxanthine and ammonium hydroxide solution (29.73%) from Fisher Sci- Thymine Uracil entific, Pittsburg, PA, USA. All purified metabolite standards Xanthine (Table 1 and Table S-1 of the Supplementary Materials) were Nucleosides obtained through Sigma-Aldrich (St. Louis, MO, USA) and are Adenosine ≥98% pure according to the manufacturer, with the exception of Cytidine guanosine 5 -diphosphate, 3 -diphosphate, which was the kind Deoxyadenosine gift of Michael Cashel, National Institute of Child Health and Deoxyguanosine Human Development, Bethesda, MD, USA. All media compo- Deoxyinosine Deoxyuridine nents (see Section 2.7), were obtained from Sigma-Aldrich and Guanosine 13 are ≥98% pure according to the manufacturer. [ C]Glucose Inosine (99%) was obtained from Cambridge Isotope Laboratories Thymidine (Andover, MA, USA). Internal standards tested were N-acetyl- Uridine glutamine (not isotope-labeled) from Sigma-Aldrich and the Xanthosine following isotope-labeled compounds (uniformly 13C > 98%, Nucleoside monophosphates 15N > 98% unless otherwise indicated): glutamate, threonine, AMP 13 CMP and succinate (1,4- C) from Cambridge Isotope Laboratories; Cyclic-AMP and alanine, deoxyadenosine, thymidine, UMP, AMP, ATP, and damp TTP from Medical Isotopes (Pelham, NH, USA). All chromatog- dCMP 78 S.U. Bajad et al. / J. Chromatogr. A 1125 (2006) 76–88 Table 1 (Continued ) Table 1 (Continued ) dGMP Phosphoenolpyruvate dTMP Trehalose dUMP UDP-d-glucose GMP UDP-d-glucuronate IMP UDP-N-acetyl-d-glucosamine UMP Vitamins and derivatives Xanthosine-5-P Biotin Nucleoside di/triphosphates Carnitine ADP Folate ATP Nicotinamide CDP Nicotinate CTP Pantothenate dATP Pyridoxine dCDP Riboflavin dCTP Thiamine dGDP 5-Methyl-THF dGTP 7,8-Dihydrofolate dUTP Thiamine-P GDP Carboxylic acids GTP Acetoacetate IDP Citrate ITP Fumarate dTDP Malate TTP Oxaloacetate UDP Succinate UTP Aconitate Nucleotide precursors/derivatives ␣-Ketoglutarate Carbamoyl-l-aspartate Redox-electron-carriers and precursors Carbamoyl-P FAD Dihydroorotate FMN Guanosine 5 -PP,3 -PP NAD+ N-Acetyl-glucosamine-1-P NADH Orotate NADP+ Orotidine-P NADPH PRPP Oxidized glutathione CoA’s Quinolinate 3-Hydroxy-3-methylglutaryl-CoA Reduced glutathione Acetoacetyl-CoA Miscellaneous metabolites Acetyl-CoA 2,3-Dihydroxybenzoate CoA Agmatine Dephospho-CoA APS Malonyl-CoA Choline Propionyl-CoA Geranyl-PP Succinyl-CoA myo-Inositol Carbohydrate derivatives/precursors p-Hydroxybenzoate 2-Dehydro-d-gluconate Putrescine 3-Phosphoglycerate Spermidine 6-Phospho-d-gluconate trans, trans-Farnesyl-PP Acetylphosphate ADP-d-glucose Metabolites that are unstable or for other reasons could not be reliably measured Allantoate using the present method are marked
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