Hydrogen–Deuterium Scrambling Based on Chemical Isotope Labeling Coupled with LC–MS: Application to Amine Metabolite Identification in Untargeted Metabolomics

Abstract

Identification of metabolites at the trace level in complex samples is still one of the major challenges in untargeted metabolomics. One formula in the metabolomic database is always corresponding to more than one candidate, which increases the difficulty and cost in the subsequent process of standard compound matching. In this study, we developed an effective method for amine metabolite identification by hydrogen–deuterium scrambling (HDS) based on chemical isotope labeling coupled with liquid chromatography–mass spectrometry (HDS–CIL–LC–MS). After d 4-4-(N,N-dimethylamino)­phenyl isothiocyanate (d 4-DMAP) labeling, the labeled amine metabolites can produce HDS under collision-induced dissociation (CID). The HDS can effectively reflect the number of labile hydrogen atoms in amine metabolites and thus distinguish amine isomers with different functional groups. The developed HDS–CIL–LC–MS method was applied to the determination of amine metabolites in mice feces, in which the amine candidates obtained by the database based on chemical formula searching were reduced by 64% on average, which greatly reduces the cost of standard compound matching. Taken together, the developed HDS–CIL–LC–MS analysis was demonstrated to be a promising method for untargeted metabolomics and a novel strategy for deciphering tandem mass spectrometry (MS2) spectra.

Oxygen Isotope Exchange Reaction for Untargeted LC–MS Analysis

“…The oxygen isotope label has an advantage over deuterium labels of being nonlabile during MS/MS fragmentation, while for hydrogen/deuterium atoms phenomena such as scrambling were observed. 28 Moreover, hydrogens are typically removed in cheminformatics software, making it challenging to process deuterium-labeled molecules. Therefore, the information gathered during MS/MS fragmentation of 18 O-labeled molecules can be used for identification.…”

Enzyme-Catalyzed Hydrogen–Deuterium Exchange between Environmental Pollutants and Enzyme-Regulated Endogenous Metabolites

“…Moreover, the rate of H–D exchange is dependent on the dynamics of a protein, particularly the stability of its hydrogen bonding networks . H–D exchange coupled with liquid chromatography–mass spectrometry has been used to identify metabolites, whereby D 2 O and CD 3 OD are used as mobile phases to ensure the exchanges of D atoms with functional groups, such as hydroxyl, carboxyl, or amide groups. , Subsequently, the fragmentation patterns of the D-labeled targeted compounds are used to identify their metabolic products. , Furthermore, H–D exchange was recently reported to occur between various chemicals upon the addition of collision energy . Specifically, amine metabolites were treated with tetradeuterated 4-( N , N -dimethylamino)­phenyl isothiocyanate to produce D-labeled amine metabolites via collision-induced dissociation .…”

Metabolic Profiling of Urinary Chiral Amino-Containing Biomarkers for Gastric Cancer Using a Sensitive Chiral Chlorine-Labeled Probe by HPLC-MS/MS

“…32,33 To address these issues, an indirect analytical method by derivatization reagents 34 has been developed and used in the clinic 35 based on LC−MS, GC− MS, and CE-MS. 36 Among them, chemical isotope labeling (CIL) in combination with LC−MS has emerged as a remarkable strategy in analysis of chiral amino-containing compounds. 35 Several CIL reagents such as (S)-COXA-OSu and (S)-COXA-d 5 -OSu, 37 16 O 2 -/ 18 O 2 -based CIL reagents, 38 12 C 2 -/ 13 C 2 -MBAA-NHS, 12 C 2 -/ 13 C 2 -DBAA-NHS, 39 DMAP, and d 4 -DMAP, 40 have been developed for the labeling of amino-containing metabolites. Another stable isotope labeling reagent example is the duplex mass defect-based N,N-dimethyl leucine (mdDiLeu) reagents, which have a mass difference of 20.5 mDa between the two DiLeu isotopologues, and they were applied to the quantitative analysis of proteins and amine metabolites in pancreatic cancer cells.…”