Chemoselective detection and discrimination of carbonyl-containing compounds in metabolite mixtures by ¹ H-detected ¹⁵ N nuclear magnetic resonance

Abstract

NMR spectra of mixtures of metabolites extracted from cells or tissues are extremely complex, reflecting the large number of compounds that are present over a wide range of concentrations. Although multidimensional NMR can greatly improve resolution as well as improve reliability of compound assignments, lower abundance metabolites often remain hidden. We have developed a carbonyl selective aminooxy probe that specifically reacts with free keto and aldehyde functions, but not carboxylates. By incorporating 15N in the aminooxy functional group, 15N-edited NMR was used to select exclusively those metabolites that contain a free carbonyl function while all other metabolites are rejected. Here we demonstrate that the chemical shifts of the aminooxy adducts of ketones and aldehydes are very different, which can be used to discriminate between aldoses and ketoses for example. Utilizing the 2 or 3 bond 15N-1H couplings, the 15N-edited NMR analysis was optimized first with authentic standards and then applied to an extract of the lung adenocarcinoma cell line A549. More than 30 carbonyl containing compounds at NMR detectable levels, 6 of which we have assigned by reference to our database. As the aminooxy probe contains a permanently charged quaternary ammonium group, the adducts are also optimized for detection by mass spectrometry. Thus, this sample preparation technique provides a better link between the two structural determination tools, thereby paving the way to faster and more reliable identification of both known and unknown metabolites directly in crude biological extracts.

Applications of NMR spectroscopy to systems biochemistry

“…Other CS probes have been developed by us that select for functional groups including thiols [35] and carbonyls (aldehydes and ketones) [36,37]. The resulting adducts in biological extracts are optimized for both MS analysis, and 15 N edited 1 H NMR analysis that makes use of long range 2 and 3-bond couplings [31]. This has been illustrated for the carbonyl-selective aminooxy probe, for which 2D 1 H{ 15 N}-HSQC spectra (cf.…”

“…This has been illustrated for the carbonyl-selective aminooxy probe, for which 2D 1 H{ 15 N}-HSQC spectra (cf. Section 4.1.4) have been acquired for assignment purpose [31]. …”

Emerging New Strategies for Successful Metabolite Identification in Metabolomics

“…This characteristic has recently been utilized to improve metabolite identification. Two 15 N labeled agents, cholamine tag [87] and aminooxy probe [88] have been introduced that selectively and covalently attach to carboxyl and carbonyl group containing metabolites, respectively. This makes signals of these metabolites directly visible in 2D 15 N-1 H HSQC NMR spectra and mass spectra because of the introduction of a permanent charge, thereby linking NMR and MS signals of the modified metabolites.…”

“…The approach does not require purifications of unknown compounds from the matrix, • Introduces 15 N-cholamine tag to facilitate detection of carboxyl group containing metabolites in NMR and MS spectra of human serum and urine. 88 Lane AN, Arumugam S, Lorkiewicz PK et al…”

“…The approach does not require purifications of unknown compounds from the matrix, • Introduces 15 N-cholamine tag to facilitate detection of carboxyl group containing metabolites in NMR and MS spectra of human serum and urine. 88 Lane AN, Arumugam S, Lorkiewicz PK et alChemoselective detection and discrimination of carbonylcontaining compounds in metabolite mixtures by 1H-detected 15N nuclear magnetic resonance. Magn.…”