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Low mass MS/MS fragments of protonated amino acids used for distinction of their 13C-isotopomers in metabolic studies.

ABSTRACT: Glu, Gln, Pro, and Ala are the main amino acids involved in ammonia detoxification in mosquitoes. In order to develop a tandem mass spectrometry method (MS(2)) to monitor each carbon of the above isotopically-labeled (13)C-amino acids for metabolic studies, the compositions and origins of atoms in fragments of the protonated amino acid should be first elucidated. Thus, various electrospray (ESI)-based MS(2) tools were employed to study the fragmentation of these unlabeled and isotopically-labeled amino acids and better understand their dissociation pathways. A broad range of fragments, including previously-undescribed low m/z fragments was revealed. The formulae of the fragments (from m/z 130 down to m/z 27) were confirmed by their accurate masses. The structures and conformations of the larger fragments of Glu were also explored by ion mobility mass spectrometry (IM-MS) and gas-phase hydrogen/deuterium exchange (HDX) experiments. It was found that some low m/z fragments (m/z 27-30) are common to Glu, Gln, Pro, and Ala. The origins of carbons in these small fragments are discussed and additional collision induced dissociation (CID) MS(2) fragmentation pathways are proposed for them. It was also found that small fragments (?m/z 84) of protonated, methylated Glu, and methylated Gln are the same as those of the underivatized Glu and Gln. Taken together, the new approach of utilizing low m/z fragments can be applied to distinguish, identify, and quantify (13)C-amino acids labeled at various positions, either in the backbone or side chain.

SUBMITTER: Ma X

PROVIDER: S-EPMC3624025 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Project description:It has become increasingly important to qualitatively and quantitatively assess the volatile metabolites in a range of bodily fluids for use in monitoring health. There has been relatively little work on the quantitative analysis of compounds, particularly with respect to the effects of ethnicity or geographic location. A novel method for the quantification of compounds in stool using 13C labelled compounds as internal standards is presented. Using thermal desorption gas chromatography mass spectrometry, stool samples from 38 healthy volunteers were analysed. The 13C labelled compounds, acetone, ethyl butanoate, ethanoic acid, butanoic acid, 3-methylbutanoic acid, and indole, were added as internal standards. This process mimics the solubility characteristics of the compounds and thus the method was able to quantify the compounds within the solid stool. In total, 15 compounds were quantified: Dimethyl sulphide (26⁻25,626 ng/g), acetone (442⁻3006 ng/g), ethyl butanoate (39⁻2468 ng/g), ethyl 2-methylbutanoate (0.3⁻180 ng/g), dimethyl disulphide (35⁻1303 ng/g), 1-octen-3-one (12 ng/g), dimethyl trisulphide (10⁻410 ng/g), 1-octen-3-ol (0.4⁻58 ng/g), ethanoic acid (672⁻12,963 ng/g), butanoic acid (2493⁻11,553 ng/g), 3-methylbutanoic acid (64⁻8262 ng/g), pentanoic acid (88⁻21,886 ng/g), indole (290⁻5477 ng/g), and 3-methyl indole (37⁻3483 ng/g). Moreover, by altering the pH of the stool to pH 13 in conjunction with the addition of 13C trimethylamine, the method was successful in detecting and quantifying trimethylamine for the first time in stool samples (range 40⁻5312 ng/g). Statistical analysis revealed that samples from U.K. origin had five significantly different compounds (ethyl butanoate, 1-octen-3-ol, ethanoic acid, butanoic acid, pentanoic acid, and indole) from those of South American origin. However, there were no significant differences between vegetarian and omnivore samples. These findings are supported by pre-existing literature evidence. Moreover, we have tentatively identified 12 compounds previously not reported as having been found in stool.

Project description:BACKGROUND:Millions of adults have been reported with hyperlipemia in the world. It is still unclear whether the plasma level of essential amino acids (AAs) will be influenced by the hyperlipemia. This study was aimed to investigate the AAs levels and the underlying metabolic relationship in hyperlipidemic subjects. METHODS:An ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed for the determination of phenylalanine (Phe), valine (Val), histidine (His), tryptophan (Trp), and methionine (Met). Plasma samples (100??L) were precipitated by acetonitrile (300??L) and analyzed on a BEH C18 (2.1?mm?×?100?mm, 1.7??m) column at 40?°C by gradient elution. The mobile phase composed of 0.1% formic acid and acetonitrile was used with flow rate at 0.2-0.4?ml/0-3?min. Five AAs were determined at positive electrospray ionization (ESI+) at m/z 118.1/72.1 (Val), 150.12/104.02(Met), 156.06/110.05(His), 166.1/120.1(Phe), and 205.2/188.02 (Trp). A total of 75 healthy subjects and 83 hyperlipidemic subjects, who had blood routine test and plasma lipid test were determined by developed UPLC-MS/MS. RESULTS:It was shown that there was good linearity for Val, Met, His, Phe, and Trp within 1-100??g/mL. The relative standard deviations of precision and accuracy were all within 15%. The level of Val, Phe, Trp, His, and Met were 35.34?±?15.64, 22.72?±?9.13, 17.23?±?4.94, 16.78?±?13.64, and 6.24?±?1.97??g/mL in healthy subjects, while they were 38.04?±?16.70, 22.41?±?8.45, 15.62?±?5.77, 18.35?±?14.49, and 6.21?±?1.97??g/mL in hyperlipidemic subjects respectively. The Spearman's correlations analysis showed that there were high correlations between Val, Phe, Trp, His, Met and triglyceride in healthy subjects. While, those correlations decreased in hyperlipemia cases. CONCLUSION:A convenient and sensitive method for simultaneous determination of Val, Phe, Trp, His, and Met in human plasma was developed. There was a high correlation between Val, Phe, Trp, His, Met and triglyceride. Hyperlipemia influences the metabolic balance of His, Phe, Trp, Met and Val.

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Low mass MS/MS fragments of protonated amino acids used for distinction of their 13C-isotopomers in metabolic studies.

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