Abstract
Metabolomics profiling using liquid chromatography-mass spectrometry (LC-MS) has become an important tool in biomedical research. However, resolving enantiomers still represents a significant challenge in the metabolomics study of complex samples. Here, we introduced N,N-dimethyl-l-cysteine (dimethylcysteine, DiCys), a chiral thiol, for the o-phthalaldehyde (OPA) derivatization of enantiomeric amine metabolites. We took interest in DiCys because of its potential for multiplex isotope-tagged quantification. Here, we characterized the usefulness of DiCys in reversed-phase LC-MS analyses of chiral metabolites, compared against five commonly used chiral thiols: N-acetyl-l-cysteine (NAC); N-acetyl-d-penicillamine (NAP); isobutyryl-l-cysteine (IBLC); N-(tert-butoxycarbonyl)-l-cysteine methyl ester (NBC); and N-(tert-butylthiocarbamoyl)-l-cysteine ethyl ester (BTCC). DiCys and IBLC showed the best overall performance in terms of chiral separation, fluorescence intensity, and ionization efficiency. For chiral separation of amino acids, DiCys/OPA also outperformed Marfey’s reagents: 1-fluoro-2-4-dinitrophenyl-5-l-valine amide (FDVA) and 1-fluoro-2-4-dinitrophenyl-5-l-alanine amide (FDAA). As proof of principle, we compared DiCys and IBLC for detecting chiral metabolites in aqueous extracts of rice. By LC–MS analyses, both methods detected twenty proteinogenic l-amino acids and seven d-amino acids (Ala, Arg, Lys, Phe, Ser, Tyr, and Val), but DiCys showed better analyte separation. We conclude that DiCys/OPA is an excellent amine-derivatization method for enantiomeric metabolite detection in LC-MS analyses.
Highlights
In the post-genomics era, metabolomics profiling has become an important tool in biomedical research [1,2,3,4]
Land D amino acids (Glu, Ser, Ala, Tyr, Phe), L -Cys, OPA, ammonium bicarbonate, perchloric acid (ACS reagent, 70%), formaldehyde (37% w/w), dichloromethane, ninhydrin, fluorescamine, Ellman’s reagent (DTNB), NAC, NAP, IBLC, N-(tert-butoxycarbonyl)-L-cysteine methyl ester (NBC), and BTCC were purchased from Sigma-Aldrich
We systematically evaluated the suitability of six chiral thiols (DiCys, NAC, NAP, IBLC, NBC, and BTCC) for OPA-assisted separation of amino acid enantiomers
Summary
In the post-genomics era, metabolomics profiling has become an important tool in biomedical research [1,2,3,4]. Initial interests in chiral metabolomics began with D-amino acids, which are physiologically active substances in mammals [16,17]. D-serine, D-aspartate, D-alanine, and D-cysteine are found in many tissues and body fluids, and several D-amino acids are important neurotransmitters in the brain [18,19]. Enantiomeric amino acids and their derivatives may be useful biomarkers and novel drug candidates; their detection is important in pharmacological research, clinical analysis, agriculture, and food science [20,21,22]
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