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. 2016 Jul 5;88(13):6658-61.
doi: 10.1021/acs.analchem.6b02009. Epub 2016 Jun 23.

Profiling Reactive Metabolites via Chemical Trapping and Targeted Mass Spectrometry

Jae Won Chang  1 , Gihoon Lee  1 , John S Coukos  1 , Raymond E Moellering  1
Affiliations

Profiling Reactive Metabolites via Chemical Trapping and Targeted Mass Spectrometry

Jae Won Chang et al. Anal Chem. .

Abstract

Metabolomic profiling studies aim to provide a comprehensive, quantitative, and dynamic portrait of the endogenous metabolites in a biological system. While contemporary technologies permit routine profiling of many metabolites, intrinsically labile metabolites are often improperly measured or omitted from studies due to unwanted chemical transformations that occur during sample preparation or mass spectrometric analysis. The primary glycolytic metabolite 1,3-bisphosphoglyceric acid (1,3-BPG) typifies this class of metabolites, and, despite its central position in metabolism, has largely eluded analysis in profiling studies. Here we take advantage of the reactive acylphosphate group in 1,3-BPG to chemically trap the metabolite with hydroxylamine during metabolite isolation, enabling quantitative analysis by targeted LC-MS/MS. This approach is compatible with complex cellular metabolome, permits specific detection of the reactive (1,3-) instead of nonreactive (2,3-) BPG isomer, and has enabled direct analysis of dynamic 1,3-BPG levels resulting from perturbations to glucose processing. These studies confirmed that standard metabolomic methods misrepresent cellular 1,3-BPG levels in response to altered glucose metabolism and underscore the potential for chemical trapping to be used for other classes of reactive metabolites.

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Figures

Figure 1
Figure 1
Hydroxylamine trapping of 1,3-BPG in vitro. (A-B) Relative aggregate1,3-BPG/2,3-BPG levels (A) and 3PG levels (B) resulting from in vitro GAPDH enzyme reactions and subsequent standard metabolomic workup and LC- MS/MS analysis. (C) Concentration-dependent formation of 3PGha by NH2OH. (D) LC-MS/MS chromatograms of 3PGha formation measured with the 199.98→79 ion transition shown above. (E) Enzymatic (Enz) and synthetic (syn) 3PGha target ion chromatograms. Data shown represent mean ± S.E.M. from triplicate experiments. Statistical significance was determined by two-way t tests: **p< 0.01,***p< 0.005.
Figure 2
Figure 2
Chemical trapping of cellular 1,3-BPG in response to altered metabolic conditions. (A) Schematic representation of an in situ trapping workflow for unstable 1,3-BPG. (B) Comparison of 3PGha levels detected in IMR32 cells as a result of altered hydroxylamine trapping methods. (C & D) Extracted ion chromatograms of aggregate BPG (C) and 3PGha (D) in NaF treated IMR32 cells. (+/−) indicates with or without NH2OH trapping. (E) Relative changes in BPG and 3PGha levels in response to NaF treatment. (F & G) Extracted ion chromatograms of aggregate BPG (F) and 3PGha (G) IMR32 cells grown in 0, 10 or 25 mM glucose. (+/−) indicates with or without NH2OH trapping. (H) Relative changes in BPG and 3PGha levels in response to different glucose concentrations. Data shown represent mean ± S.E.M. from triplicate experiments. Statistical significance was determined by two-way t tests: **p< 0.01, ***p< 0.005.
Scheme 1
Scheme 1
Chemical trapping of 1,3-BPG and competing reactions in central glucose metabolism.
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