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Methylation is an essential metabolic process in the biological systems, and it is significant for several biological reactions in living organisms. Methylated compounds are known to be involved in most of the bodily functions, and some of them serve as biomarkers. Theoretically, all α-amino acids can be methylated, and it is possible to encounter them in most animal/plant samples. But the analytical data, especially the mass spectral data, are available only for a few of the methylated amino acids. Thus, it is essential to generate mass spectral data and to develop mass spectrometry methods for the identification of all possible methylated amino acids for future metabolomic studies. In this study, all N-methyl and N,N-dimethyl amino acids were synthesized by the methylation of α-amino acids and characterized by a GC-MS method. The methylated amino acids were derivatized with ethyl chloroformate and analyzed by GC-MS under EI and methane/CI conditions. The EI mass spectra of ethyl chloroformate derivatives of N-methyl (1-18) and N,N-dimethyl amino acids (19-35) showed abundant [M-COOC 2 H 5 ] + ions. The fragment ions due to loss of C 2 H 4 , CO 2 , (CO 2  + C 2 H 4 ) from [M-COOC 2 H 5 ] + were of structure indicative for 1-18. The EI spectra of 19-35 showed less number of fragment ions when compared with those of 1-18. The side chain group (R) caused specific fragment ions characteristic to its structure. The methane/CI spectra of the studied compounds showed [M + H] + ions to substantiate their molecular weights. The detected EI fragment ions were characteristic of the structure that made easy identification of the studied compounds, including isomeric/isobaric compounds. Fragmentation patterns of the studied compounds (1-35) were confirmed by high-resolution mass spectra data and further substantiated by the data obtained from 13 C 2 -labeled glycines and N-ethoxycarbonyl methoxy esters. The method was applied to human plasma samples for the identification of amino acids and methylated amino acids. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Methylation is an essential metabolic process for a number of critical reactions in the body. Methyl groups are involved in the healthy function of the body life processes, by conducting methylation process involving specific enzymes. In these processes, various amino acids are methylated, and the occurrence of methylated amino acids in nature is diverse. Nowadays, mass-spectrometric-based identification of small molecules as biomarkers for diseases is a growing research. Although all dimethyl amino acids are metabolically important molecules, mass spectral data are available only for a few of them in the literature. In this study, we report synthesis and characterization of all dimethyl amino acids, by electrospray ionization-tandem mass spectrometry (MS/MS) experiments on protonated molecules. The MS/MS spectra of all the studied dimethyl amino acids showed preliminary loss of H2O + CO to form corresponding immonium ions. The other product ions in the spectra are highly characteristic of the methyl groups on the nitrogen and side chain of the amino acids. The amino acids, which are isomeric and isobaric with the studied dimethyl amino acids, gave distinctive MS/MS spectra. The study also included MS/MS analysis of immonium ions of dimethyl amino acids that provide information on side chain structure, and it is further tested to determine the N-terminal amino acid of the peptides., (Copyright © 2015 John Wiley & Sons, Ltd.)

Betaines belong to the naturally occurring osmoprotectants or compatible solutes present in a variety of plants, animals and microorganisms. In recent years, metabolomic techniques have been emerging as a fundamental tool for biologists because the constellation of these molecules and their relative proportions provide with information about the actual biochemical condition of a biological system. Therefore, identification and characterization of biologically important betaines are crucial, especially for metabolomic studies. Most of the natural betaines are derived from amino acids and related homologues. Although, theoretically, all the amino acids can be converted to corresponding betaines by simple methylation of the amine group, only a few of the amino acid-derived betaines were fully characterized in the literature. Here, we report a combined electrospray ionization tandem and high-resolution mass spectrometry study of all the betaines derived from amino acids, including the isomeric betaines. The decomposition pathway of protonated, sodiated and potassiated molecule ions that enable unambiguous characterization of the betaines including the isomeric betaines and overlapping ionic species of different betaines is distinctive., (Copyright © 2012 John Wiley & Sons, Ltd.)

A set of diastereomeric α-sulfanyl-β-amino acid derivatives, which are important building blocks for pharmaceuticals with potent biological activity, are studied by electrospray ionization tandem mass spectrometry. The collision induced dissociation (CID) spectra of [M+H](+), [M+NH(4)](+), [M+Na](+) and [M+Li](+) of the diastereomers were studied, among them the CID of [M+Na](+) and [M+Li](+) showed consistent differences in the relative abundance of characteristic ions that enabled distinction of the anti isomers from syn isomers. The decomposition pathways for the diagnostic ions were arrived at based on high-resolution mass spectrometry data, multiple mass spectrometry data, deuterium labeling experiments and the mass shift in accordance with the substituents located at different places. Loss of (R(1)-C(6)H(4)-CH=NH) and (Cat-NH-SO(2)R(2)) from [M+Cat](+), where Cat=Na and Li, and the product ions as a results of McLafferty rearrangement involving either >S=O or >C=O group were found to be diagnostic. The McLafferty rearrangement product ions involving >S=O group were more abundant in syn isomers while those involving >C=O group were more abundant in anti isomer. The selectivity observed in the decomposition of [M+Li](+) ions was found to be similar to that of [M+Na](+) ions, but in few cases the differences are marginal in the decomposition [M+Li](+) ions.

Halogen-substituted phenylalanines with a halogen X (X = F, Cl, Br or I) in the para position in the aromatic ring of L-phenylalanine are used as enantioselective selectors to explore the effect of the halogen substituent on the enantioselective discrimination of amino acids. Enantioselective discrimination is achieved by investigating the collision-induced dissociation spectra of the trimeric complex ion, [CuII(ref)2(A)-H]+, generated by electrospraying a solution of a mixture of D- or L-analyte amino acid (A), enantioselective reference ligand (ref) and CuCl2. The relative abundances of fragment ions resulting from the competitive loss of reference and analyte amino acids are considered for measuring the degree of enantioselective discrimination by applying the kinetic method. The enantioselectivity of the p-halogenated derivatives of L-Phe increases from fluorine to iodine for the studied amino acids (except for acidic amino acids). The validity of the present method has also been checked by cross enantioselective experiments using p-iodo-D-phenylalanine as the reference in place of p-iodo-L-phenylalanine. The enantioselectivity of fluoro-substituted L-phenylalanine is less than that obtained with L-phenylalanine. The high inductive effect of the fluorine atom decreases the strength of the pi-pi stacking interaction. The presence of halogen affects the enantioselectivity by inductive and steric effects., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

A set of DNA tetranucleotides, which are 3'- or 5'-end extended versions of GCA, was used as chiral selectors for the discrimination of enantiomers of alpha-amino acids. The [X+Y-2H](2-) ions of the 1:1 complexes were generated by electrospraying a mixture of tetranucleotide (X) and amino acid (Y) solution. Chiral discrimination was achieved by studying the collision-induced dissociation spectra of the [X+Y-2H](2-) ion and the ratio of relative abundance of precursor ion to that of the product ion was used to measure the extent of discrimination. Among the tetranucleotides used, GCAA and GGCA exhibited better discrimination, in which GCAA showed D-selectivity and GGCA showed L-selectivity for the studied amino acids. In addition, binding constants were measured for the 1:1 complexes of phenylalanine enantiomers with GCAA and GGCA. Ltd., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

L-Tyrosine and iodinated L-tyrosines, i.e., 3-iodo-L-tyrosine and 3,5-diiodo-L-tyrosine, are successfully used as chiral references for the chiral discrimination of aliphatic, acidic, and aromatic amino acids. Chiral discrimination is achieved by investigating the collision-induced dissociation spectra of the trimeric complex [Cu(II)(ref)(2)(A) - H](+) ion generated by electro spraying the mixture of D- or L-analyte amino acid (A), chiral reference ligand (ref) and M(II)Cl(2) (M = Ni and Cu). The relative abundances of fragment ions resulted by the competitive loss of reference and analyte amino acids are considered for measuring the degree of chiral discrimination by applying the kinetic method. The chiral discrimination ability increases as the number of iodine atom increases on the aromatic ring of the reference and the discrimination is better with Cu when compared with Ni. A large chiral discrimination is obtained for aliphatic and aromatic amino acids using iodinated L-tyrosine as the reference. Computational studies on the different stabilities of the diastereomeric complexes also support the observed differences measured by the kinetic method. The suitability of the method in the measurement of enantiomeric excess over the range of 2% to 100% ee with relative error 0.28% to 1.6% is also demonstrated.

The DNA triplet GCA is successfully used for the first time as a chiral selector for the chiral discrimination and optical purity measurement of some alpha-amino acids by investigating the collision-induced dissociation spectra of the sodiated ternary complex ion formed by electrospray ionization.

Hereditary xerocytosis (HX), also known as dehydrated stomatocytosis (DHSt) is a dominantly inherited genetic disorder exhibiting red cell membrane dehydration caused by the loss of the monovalent cation K+ and water. Variants in mechanosensitive Piezo ionic channels of the PIEZO1 gene are the primary cause of HX. We have utilized high throughput and highly precise next-generation sequencing (NGS) to make a diagnosis and examine the genotype-phenotype relationship in inflexible HX cases. Seven unrelated patients with unexplained hemolytic anemia were scrutinized with a panel probing 8000 genes related to congenital anemia. Targeted next-generation sequencing identified 8 missense variants in the PIEZO1 gene in 7 unrelated Indian patients. Three of the 8 variants are novel (c.1795G > C, c.2915G > A, c.7372 T > C) and the remaining five (c.4082A > G, c.6829C > A, c.7374C > G, c.7381G > A, c.7483_7488dup) are previously reported. The variants have been validated by Sanger sequencing. One patient with autosomal dominant mutation (c.7372 T > C) is associated with iron refractory iron deficiency anemia. Of the 7 patients, one has HX in combination with a novel homozygous variant (c.994G > A) in the PKLR gene causing PK deficiency resulting in severe clinical manifestations with phenotypic variability. In silico prediction using bioinformatics tools were used to study the possible damaging effects of the novel variants. Structural-functional analysis of the novel variants was investigated by molecular modeling software (PyMOL and Swiss PDB). These results encompass the heterogeneous behavior of mechano-sensitive Piezo1 protein observed in HX patients in India. Moreover, NGS imparted a subtle, economical, and quick tool for understanding the genetic cause of undiagnosed cases of congenital hemolytic anemia. NGS grants a potential technology integrating clinical history together with molecular report profiting in such patients and their families. [ABSTRACT FROM AUTHOR]

Rationale: Methylglyoxal (MGO) and glyoxal (GO) are known to be at high levels in humans with diabetes. They react with amine‐containing proteins and amino acids to form advanced glycation end products, however, their reactivity with other amine‐containing metabolites, such as neurotransmitters, has not been explored. In this study, we aimed at studying the reactivity of 5‐hydroxytryptamine (5‐HT) with MGO or GO, which may alter the metabolic function of 5‐HT. Methods: Stock solutions of 5‐HT, MGO and GO were made in PBS buffer at pH 7.4 and 5‐HT was incubated with MGO or GO at different concentrations. The reactions were also performed at physiological concentrations. The reaction mixtures collected at different incubation times were analyzed by direct ESI‐HRMS, LC/MS and LC/MS/MS to detect/characterize the products. Agilent 6545 Q‐TOF and Agilent 6420 triple quadrupole mass spectrometers were used for the study, and LC separations were performed on a C18 column. Results: The direct ESI‐HRMS data of the reaction mixtures showed formation of three and four reaction products when 5‐HT was reacted with MGO and GO, respectively. All the products showed dominant [M + H]+ ions. The products were characterized by HRMS, LC/MS/MS and literature reports on similar compounds. The products can easily be identified by LC/MS based on the accurate mass values together with retention time information. The MS/MS of the reaction products showed structure‐indicative fragment ions. Conclusions: 5‐HT reacts with one or two MGO/GO to form a set of reaction products. The reaction between 5‐HT and MGO or GO was faster at higher concentrations of MGO/GO (<10 min), and the same products were found even at physiological concentrations (<48 h). The LC/MS/MS (SRM) method can be used to screen the reaction products when present at low levels. [ABSTRACT FROM AUTHOR]

Aberrant activation of the three-amino-acid-loop extension homeobox gene MEIS1 shortens the latency and accelerates the onset and progression of acute leukemia, yet the molecular mechanism underlying persistent activation of the MEIS1 gene in leukemia remains poorly understood. Here we used a combined comparative genomics analysis and an in vivo transgenic zebrafish assay to identify six regulatory DNA elements that are able to direct green fluorescent protein expression in a spatiotemporal manner during zebrafish embryonic hematopoiesis. Analysis of chromatin characteristics and regulatory signatures suggests that many of these predicted elements are potential enhancers in mammalian hematopoiesis. Strikingly, one of the enhancer elements (E9) is a frequent integration site in retroviral-induced mouse acute leukemia. The genomic region corresponding to enhancer E9 is differentially marked by H3K4 monomethylation and H3K27 acetylation, hallmarks of active enhancers, in multiple leukemia cell lines. Decreased enrichment of these histone marks is associated with downregulation of MEIS1 expression during hematopoietic differentiation. Further, MEIS1/HOXA9 transactivate this enhancer via a conserved binding motif in vitro, and participate in an autoregulatory loop that modulates MEIS1 expression in vivo. Our results suggest that an intronic enhancer regulates the expression of MEIS1 in hematopoiesis and contributes to its aberrant expression in acute leukemia. [ABSTRACT FROM AUTHOR]

Contents The present study aims to ascertain the influence of γ-amino butyric acid (GABA)A or B receptors on arginine vasopressin (AVP) release in vitro and determine whether E2 modulates GABA–AVP interaction. Within 10 min of ewe killing, saggital midline hypothalamic slices (from the anterior preoptic area to the mediobasal hypothalamus along with the median eminence, 2-mm thick, two per ewe) were dissected, placed in oxygenated minimum essential media (MEM)- α at 4°C and within 2 h were singly perifused at 37°C with oxygenated MEM- α (pH 7.4; flow rate 0.15 ml/min), either with or without E2 (24 pg/ml). After 4-h equilibration, 10-min fractions were collected for 4 h interposed with a 10-min exposure at 60 min to a specific GABAA or B receptor agonist or antagonist at various doses (0.1–10 mm). GABAA (muscimol; no E2, n = 7 perifusion chambers, with E2, n = 11) or GABAB (baclofen; no E2, n = 8, with E2, n = 15) agonists (10 mm) did not influence AVP concentrations. However, AVP release increased (p < 0.05) 20–30 min after exposure to 10 mm GABAA or B antagonists (bicuculline, no E2, n = 7: from 4.6 ± 0.7 to 33.0 ± 0.4, with E2, n = 17: from 11.9 ± 1.4 to 32.8 ± 6.0; CGP52432, with E2, n = 14: from 14.0 ± 2.6 to 28.8 ± 3.9 pg/ml). At the end of the collection period, hypothalamic slices responded to KCl (100 mm) with AVP efflux (p < 0.05). GABAB but not GABAA antagonist-stimulated AVP release was enhanced in the presence of E2. In summary, AVP release is under the inhibitory influence of GABA input with further potentiation by E2 through GABAB receptors in vitro. [ABSTRACT FROM AUTHOR]

A simple method to differentiate underivatized diastereomeric hexosamine monosaccharides, glucosamine, galactosamine, and mannosamine is reported by applying the kinetic method using N-acetylhexosamines or naturally occurring amino acids as reference bases under electrospray ionization conditions. The observed differences to distinguish the diastereomeric hexosamines are found mainly due to the proton affinity (PA) differences between the analyte and the reference base. The PA values of the hexosamines are not available in the literature, and hence, we estimated them by the kinetic method using N-acetylhexosamines as reference bases. The determined PA values are 223.97 kcal/mol for glucosamine, 224.99 kcal/mol for mannosamine, and 224.71 kcal/mol for galactosamine. The similar PA values were also obtained by using amino adds as reference bases. We have applied the same methodology to quantify these hexosamines in a mixture following the three-point calibration method suggested in the literature. [ABSTRACT FROM AUTHOR]

The kinetic method for measuring proton affinities (PA) and gas-phase basicities (GB) was applied to the methyl esters of simple amino acids. The experiments show that the GB and PA values for deuterium labeled glycine methyl ester are indeed greater than that of the corresponding unlabelled glycine methyl ester. The PA of l-Ala-OCD3 is also slightly greater than that of the unlabeled alanine methyl ester. The secondary isotope effects originate, as shown by density functional theory, in differences in zero-point energies and thermal-energy corrections between H and D-bearing molecules. [Copyright &y& Elsevier]