Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry

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Folate metabolism, which is responsible for one-carbon transfer reactions in critical cellular processes including thymidine biosynthesis, is among the most important targets of antibiotic and anticancer drugs. Analysis of intracellular folates is complicated by three different types of folate modification: oxidation/reduction, methylation, and polyglutamylation. Here we present a method for quantifying the full diversity of intracellular folates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method begins with folate extraction using Ϫ75°C methanol:water, with ascorbic acid and ammonium acetate added to prevent folate interconversion. The extract is then separated using hydrophilic interaction chromatography with an amino column, ionized by positive mode electrospray, and analyzed on a triple quadrupole instrument using multiple reaction monitoring. The method has been used to profile the folate pools in Escherichia coli and Saccharomyces cerevisiae, with absolute levels of selected folates in E. coli measured by spiking extracts of cells fed uniformly 13 C-glucose with purified, unlabeled folate standards. An isotope-ratio-based approach has been applied to study the effects of trimethoprim, a clinically important antibiotic that blocks bacterial dihydrofolate reductase. In addition to causing the expected increase in oxidized and decrease in reduced folates, trimethoprim triggered a dramatic and previously unrecognized shift towards shorter polyglutamate chain lengths. This finding highlights the potential for analysis of the full spectrum of cellular folates by MS/MS to unveil novel biological phenomena. (J Am Soc Mass Spectrom 2007, 18, 898 -909)

Section: Discussionmentioning

confidence: 99%

“…We previously reported the detection of monoglutamated folate and derivatives such as 5-methyltetrahydrofolate in positive ionization mode [11]. Here we extend this approach to polyglutamated folates, presenting a systematically validated method using HILIC combined with positive ionization tandem mass spectrometry.…”

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confidence: 98%

Isotope ratio-based profiling of microbial folates

Kwon

Rabinowitz

2007

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“…The wide range of chromatography (LC) options allow physical separation of most molecules to reduce signal suppression [16]. While reversephase methods tend to utilize C18 columns, a wide range of normal phase materials are utilized [16,17] e.g., popular new materials, including hydrophilic interaction chromatography (HILIC) and aqueous normal phase (ANP) have recently been developed for improved separation of polar metabolites [18].…”

Section: Lc/ms Based Metabolomicsmentioning

confidence: 99%

Dealing with the unknown: Metabolomics and Metabolite Atlases

Bowen

Northen

2010

182

158

Metabolomics is the comprehensive profiling of the small molecule composition of a biological sample. Since metabolites are often the indirect products of gene expression, this approach is being used to provide new insights into a variety of biological systems (clinical, bioenergy, etc.). A grand challenge for metabolomics is the complexity of the data, which often include many experimental artifacts. This is compounded by the tremendous chemical diversity of metabolites. Identification of each uncharacterized metabolite is in many ways its own puzzle (compared with proteomics, which is based on predictable fragmentation patterns of polypeptides). Therefore, effective data reduction/prioritization strategies are critical for this rapidly developing field. Here we review liquid chromatography electrospray ionization mass spectrometry (LC/MS)-based metabolomics, methods for feature finding/prioritization, approaches for identifying unknown metabolites, and construction of method specific 'Metabolite Atlases'.(J Am Soc Mass Spectrom 2010, 21, 1471-1476) © 2010 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry N ature utilizes a tremendous diversity of metabolites, and it is estimated that there are Ͼ200,000 plant metabolites alone [1]. Metabolomics is a rapidly growing field for studying the small molecule composition of a biological system. Liquid chromatography coupled to electrospray ionization mass spectrometry (LC/MS) is becoming a method of choice for profiling metabolites in complex biological samples. This is due to its ability to effectively ionize a breath of metabolites with minimal fragmentation (versus gas chromatography-mass spectroscopy (GC/MS), robustness, and ability to scale-up to support tandem mass spectrometry based structural studies (versus capillary electrophoresis-mass spectrometry (CE-MS)) [2]. However, known metabolites are typically a small portion of the data obtained in a LC/MS metabolomics experiment (ϳ10%) [3] where the bulk are MS-artifacts and uncharacterized metabolites.Identification of unknown metabolites is cost and effort intensive, often requiring preparative scale isolation for nuclear magnetic resonance (NMR) studies or extensive chemical synthesis to enable structural comparisons using tandem mass spectrometry (MS/MS) [4]. Therefore, it is not surprising that the majority of reports focus on changes in metabolites that have authentic standards or at a minimum are found in metabolite databases. Yet, there are only a few thousand commercially available analytical standards due to lack of demand and the inherent instability of many metabolites. These standards and the endogenous metabolites contained in databases represent only a portion of endogenous metabolites. Therefore, effective methods for prioritizing, studying, and ultimately identifying uncharacterized metabolites is a critical development for LC/MS based metabolomics [5][6][7][8][9][10]. Here we discuss current LC/MS metabolomic approaches (detailed elsewhere [2,4,11,12]) and their inte...

“…MS/MS involving collision-activated dissociation (CAD) has been used to obtain structural information about phosphate-containing metabolites [17][18][19][20][22][23][24]. In such experiments, negative-mode ionization is generally preferred due to the acidic character of phosphate groups [17][18][19][20][22][23][24][25]. Negative-mode CAD of phosphate-containing metabolites often results in limited fragmentation, possibly due to the absence of a mobile proton [26], and thereby provides limited structural information because it mainly results in cleavages at phosphate groups, or charge-directed fragmentation [18,19,23].…”

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confidence: 99%

“…Tandem mass spectrometry (MS/MS) and exact mass measurements of intracellular metabolites have led to the identification of metabolites for improved understanding of biological pathways and discovery of potential biomarkers [17][18][19][20][21]. MS/MS involving collision-activated dissociation (CAD) has been used to obtain structural information about phosphate-containing metabolites [17][18][19][20][22][23][24]. In such experiments, negative-mode ionization is generally preferred due to the acidic character of phosphate groups [17][18][19][20][22][23][24][25].…”

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confidence: 99%

Characterization of phosphate-containing metabolites by calcium adduction and electron capture dissociation

Liu

Yoo

Håkansson

2008

Several phosphate-containing metabolites, including nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), adenosine 5=-diphosphate ribose (ADP-r), adenosine 5=-triphosphate (ATP), and guanosine 5=-triphosphate (GTP), have been characterized with electron capture dissociation (ECD) and sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) tandem mass spectrometry (MS/MS) in positive-ion mode. Calcium complexation was used to successfully produce abundant doubly charged cationic precursor ions with or without hydration. This approach enabled application of ECD to acidic metabolites for the first time. Fragmentation pathways observed in ECD and SORI-CAD of calcium-adducted phosphate-containing metabolites were complementary. Unique fragmentation was observed in ECD compared to SORI-CAD MS/MS, including ribose cross-ring cleavage for NAD and NADP, and generation of hydrated product ions, including cross-ring fragments, for hydrated ATP and GTP. A combination of ECD and CAD appears promising for maximizing structural information about metabolites. Due to the wide range of metabolites in a metabolic network, and their chemical diversity, it is highly challenging to establish analytical tools for identifying and quantifying all of them. Although nuclear magnetic resonance, Fourier transform infrared, and Raman spectroscopies are widely used for metabolite analysis [5, 9 -13], mass spectrometry (MS) has emerged as a more suitable technology for measurement of metabolites because of its wide dynamic range (10 4 -10 6 ), good sensitivity (nM), and ability to detect a diverse number of molecular species [14 -16].Phosphate-containing metabolites are mainly involved in the central carbon metabolism, which consists of glycolysis, the pentose-phosphate pathway, the tricarboxylic acid cycle, and their corresponding cofactors. Tandem mass spectrometry (MS/MS) and exact mass measurements of intracellular metabolites have led to the identification of metabolites for improved understanding of biological pathways and discovery of potential biomarkers [17][18][19][20][21]. MS/MS involving collision-activated dissociation (CAD) has been used to obtain structural information about phosphate-containing metabolites [17][18][19][20][22][23][24]. In such experiments, negative-mode ionization is generally preferred due to the acidic character of phosphate groups [17][18][19][20][22][23][24][25]. Negative-mode CAD of phosphate-containing metabolites often results in limited fragmentation, possibly due to the absence of a mobile proton [26], and thereby provides limited structural information because it mainly results in cleavages at phosphate groups, or charge-directed fragmentation [18,19,23]. However, Gross and coworkers have recently shown that highenergy CAD of metabolite anions in a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer allows differentiation between isomeric metabolites such as adenosine 5=-triphosphate (ATP) and 2=-deoxyguano...