Identification of the electrophoretic peaks of the phenylthiohydantoin derivatives of amino acids

Two techniques for identifying the peaks of phenylthiohydantoin (PTH) amino acids separated under the conditions of micellar electrokinetic chromatography were compared. The first technique is linear regression analysis, in which the retention time of an amino acid is a function of the retention times of two retention-time standards. The second technique takes into account hydrophobicity constants logD′, which were calculated using the ACD/LC Simulator 8.0 program package from ACDLabs (Canada). These constants provide an opportunity to calculate the relative migration times of PTH amino acids taking into account the velocity of the electroosmotic flow. The first technique allows us to identify the electrophoretic peaks of all 16 amino acids separated; the second procedure allows us to predict the elution order of the electrophoretic peaks; the use of a correlation equation gives better results.     

A Computer Program for Identifying Anomalous Peaks in Automated Amino Acid Analyses

Abstract

In automated ion-exchange chromatography of amino acids utilizing spectrophotometric measurement., the ratio of the areas under the 40 mμ; and 570 mμ; absorption peak tracings has been shown to be specific for each amino acid. Symmetrical, but impure peaks have ratios deviating from these norms. A simple computer program for the rapid identification of these anomalous peaks has been designed. It is proposed as an addendum to any computer program now in use with amino-acid analyzers.     

Metabolomics‐based approach for ranking the candidate structures of unidentified peaks in capillary electrophoresis time‐of‐flight mass spectrometry

One of the technical challenges encountered during metabolomics research is determining the chemical structures of unidentified peaks. We have developed a metabolomics‐based chemoinformatics approach for ranking the candidate structures of unidentified peaks. Our approach uses information about the known metabolites detected in samples containing unidentified peaks and involves three discrete steps. The first step involves identifying “precursor/product metabolites” as potential reactants or products derived from the unidentified peaks. In the second step, candidate structures for the unidentified peak are searched against the PubChem database using a molecular formula. These structures are then ranked by structural similarity against precursor/product metabolites and candidate structures. In the third step, the migration time is predicted to refine the candidate structures. Two simulation studies were conducted to highlight the efficacy of our approach, including the use of 20 proteinogenic amino acids as pseudo‐unidentified peaks, and leave‐one‐out experiments for all of the annotated metabolites with and without filtering against the Human Metabolome Database. We also applied our approach to two unidentified peaks in a urine sample, which were identified as glycocyamidine and N ‐acetylglycine. These results suggest that our approach could be used to identify unidentified peaks during metabolomics analysis.     

Discriminating Amino Resin Paints From Alkyd Resin Paints with Two Kinds of Pigments in Automotive Coatings in Forensic Analysis by FTIR Spectroscopy

The analysis of automotive coatings is important to forensic scientists, especially in the investigation of hit-and-run accidents. Amino resin paints, alkyd resin paints, and polyurethane paints are all popular automotive coatings. In this study, FTIR was employed to investigate these coatings, particular in amino resin paints. IR spectra were tentatively interpreted. The indicative peaks distinguishing amino resin paints (1550 cm^?1) and alkyd resin paints (1600 cm^?1/1580 cm^?1) were summarized. Two kinds of alkyd resin paints (with the Pigment Scarlet Powder and with the Toluidine Red), which were frequently confronted in cases and might readily be read as amino resin paints in IR spectra, were studied and interpreted. The indicative peaks (1619 cm^?1, 776 cm^?1 and 1674 cm^?1, 1494 cm^?1) were selected to discriminate these two kinds of alkyd resin paints from amino resin paints and avoid an incorrect certificate of authenticity. The data in this study can help the forensic scientists identify these three paints accurately, especially in the cases with the interference of the pigments.     

Quantitative analysis of fluorimated ethylchloroformate derivatives of non-protein amino acids using positive and negative chemical ionization gas chromatography-mass spectometry

The GC-MS characterization of the ethylchloroformate derivatives of amino acids in an aqueous medium has been applied to non-protein amino acids. Derivatization of non-protein amino acids using ethylchloroformate, trifluoroethanol, and pyridine produced strong [M + 1]⁺ and [M - 1]⁻ ions in positive and negative chemical ionization (CI) modes, respectively. Twenty-one out of the twenty-three non-protein amino acids studied produced detectable ion chromatograms in both ionization modes when methane was used as the CI reagent gas. Mass spectra of these non-protein amino acid derivatives showed characteristic [M - 19]⁺, [M + 1]⁺, [M + 29]⁺, and [M + 41]⁺ peaks in the positive chemical ionization mode, and [M - 1]⁻, and [M + 35]⁻ peaks in the negative chemical ionization mode. The detection limits and the linear dynamic range of trifluorethanol ethylchloroformate derivatives of non-protein amino acids were studied using positive chemical ionization. The detection limits are mostly in the femtomole range.     

Gas chromatographic-mass spectrometric analysis of N-acetylated amino acids: the first case of aminoacylase I deficiency

During the metabolic work-up of a patient presenting with neonatal convulsions, we consistently observed the presence of unusual peaks in the gas chromatographic-mass spectrometric analysis of urinary organic acids. The gas chromatographic-mass spectrometric characteristics of the unusual peaks suggested that they corresponded to derivatives of N-acetylated amino acids. The tentative identification was confirmed by the identity of retention times and mass spectra of the trimethylsilyl derivatives of the authentic compounds. We describe our observations that led to the identification of the various N-acetylated amino acids in this first patient with a confirmed deficiency of aminoacylase I, an enzyme involved in the cytosolic degradation of N-terminally modified proteins. The potential and limitations of urinary organic acid analysis for the detection of N-acetylated amino acids was further studied using pure compounds. In addition, we provide mass spectral data for 37 trimethylsilyl derivatives from 17 N-acetylated amino acids, most of which have not been reported previously. Our data provide valuable information that will help the clinical laboratorians who are responsible for organic acid analysis to recognize this new condition and could aid its detection in other patients.     

Determination of amino acids in overlapped capillary electrophoresis peaks by means of partial least-squares regression

Amino acid derivatives of 1,2-naphthoquinone-4-sulfonate (NQS) can be separated by capillary electrophoresis at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate-isopropanol (3:1, v/v) solution as background electrolyte. This procedure was suitable for the most common amino acids. However, the peaks of three amino acids (phenylalanine, isoleucine and tyrosine) were only partially resolved and peaks of histidine and leucine derivatives overlapped completely. Partial least-squares regression (PLS) may overcome the lack of selectivity for these amino acids. Spectroelectropherograms of the corresponding amino acid derivative peaks were monitored with a diode-array spectrophotometer in the range 225 to 540 nm. Both spectra and electropherograms can be used as multivariate data for further analysis. In general, the best predictions were obtained using the time domain.     

The emission thermophotometry (ETP) of some amino acids

The emission thermophotometry (ETP) curves of ten selected amino acids were determined in a flowing oxygen atmosphere. Most of the ETP curves contained a single emission peak in the 230–350°C temperature range; several of the curves contained multiple peaks and/or shoulder peaks. The light emission varied in intensity for each amino acid, but in most cases was at a very low level. The origin of the light emission process is not known but it is probably due to the presence of unstable pyrolysis products.     

Silylgruppenwanderung und CO2-Eliminierung beim massenspektrometrischen Zerfall von N-Trifluoracetyl-α-aminosäretrimethylsilylestern

The molecular ions of N-trifluoroacetyl α-amino acid trimethylsilyl esters exhibit a characteristic elimination of CO₂, in contrast to other amino acid derivatives and apparently caused by migration of the ester trimethylsilyl group to the oxygen atom of the N-trifluoroacetyl function. Fragmentation of the [M – CO₂]⁺˙ ions gives rise to a series of intense peaks, especially for the aliphatic amino acid derivatives. In the case of the isomers leucine and isoleucine, different base peaks are formed for the 20 eV spectra. Amino acids which can easily split off a group in their β-position possibly fragment by synchronous elimination of CO₂ and this group. With serine, threonine and cysteine a concurrent ester silyl migration to the oxygen of the β-function is observed, accompanied by the expulsion of CO₂.     

天然氨基酸水溶液中聚吡咯的电化学合成及其表征

报道了各种天然α-氨基酸水溶液中电化学聚合吡咯获得氨基酸掺杂的聚吡咯.实验表明吡咯在酸性氨基酸电解质中的氧化聚合电位较低,速度较快;而在碱性氨基酸水溶液中几乎无法进行电化学聚合.在电化学聚合过程中,氨基酸既作为支持电解质,又作为对离子被掺杂到聚合物中.该聚吡咯的电导率被测定为0.3～1.0 S/cm,在酸性氨基酸溶液中得到的聚合物电导率明显高于酸性较弱的氨基酸溶液中得到的聚合物,同时聚合物还具有良好的电化学活性和电化学稳定性,在-0.5 V到+0.5 V区间有一对氧化还原峰,该氧化还原峰的形状和特性在100次循环后基本保持不变.通过扫描电镜和透射电镜照片可以看出,不同种氨基酸的掺杂对聚吡咯的形貌具有影响,由于氨基酸的软模板效应,在数种氨基酸水溶液中能制得具有纳米纤维结构的聚吡咯.     

Quantification of glycated N‐terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry

A novel method of amino acid analysis using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups) was applied to measure glycated amino acids in order to quantify glycated peptides and evaluate the degree of glycation of peptide. Amino and carboxyl groups of amino acids were derivatized with 1‐bromobutane so that the hydrophobicities and basicities of the amino acids, including glycated amino acids, were improved. These derivatized amino acids could be detected with high sensitivity using LC‐MS/MS. In this study, 1‐deoxyfructosyl‐VHLTPE and VHLTPE, which are N‐terminal peptides of the β‐chains of hemoglobin, were selected as target compounds. After reducing the peptide sample solution with sodium borohydride, the obtained peptides were hydrolyzed with hydrochloric acid. The released amino acids were then derivatized with 1‐bromobutane and analyzed with LC‐MS/MS. The derivatized amino acids, including glycated amino acids, could be separated using an octadecyl silylated silica column and good sharp peaks were detected. We show a confirmatory experiment that the proposed method can be applied to evaluate the degree of glycation of peptides, using mixtures of glycated and non‐glycated peptide. Copyright © 2015 John Wiley & Sons, Ltd.     

Vibrational and Thermodynamic Properties of 2,2',4,4',6,6'-Hexanitroazobenzene and Its Derivatives: A Density Functional Theory Study

HNAB (2,2′,4,4′,6,6′‐hexanitroazobenzene) and its derivatives have been optimized to obtain their molecular geometries and electronic structures by using density functional theory at the B3LYP/6‐31G* level. Their IR spectra have been computed and assigned by vibrational analysis. The strongest peaks are attributed to the N? O asymmetric stretching of nitro groups. Its central position moves towards higher frequency as the number of nitro groups increases. It is obvious that there is hydrogen‐bonding between amino and nitro groups in amino derivatives. Based on the frequencies scaled by 0.96 and the principle of statistical thermodynamics, the thermodynamic properties have been evaluated, which are linearly related with the temperature, as well as the number of nitro and amino groups, respectively, obviously showing good group additivity. And the thermodynamic functions for the nitro derivatives increase much more than those for the amino derivatives with the increase of the number of substituents. The values of heat of formation (HOF) for the nitro derivatives increase gradually with n, while those of the amino derivatives decrease smoothly with n.     

Monitoring of Protease-Catalyzed Peptide Synthesis by High Performance Liquid Chromatography

Abstract

High performance liquid chromatography (HPLC) on prepacked silica gel columns has been applied to the separation of closely related protected peptides and amino acids. In the course of a protease-catalyzed synthesis of Leu-enkephalin this chromatogra-phic technique was found to be a valuable tool to rapidly and reliably characterize the outcome of enzymatic reactions, the nature of which was often difficult to be predicted. Stepwise gradient elution was employed to enable fractionation of mixture components, which covered only a short polarity range. The solvent systems composed of dichloromethane, anhydrous ethanol and acetic acid were mixed in such ratios so as to provide completely resolved peaks for the sample problems studied so far. Enzymatically prepared compounds and their chemically synthesized, authentic analogues were cochromatographed to enable the assignment of the eluted peaks. The provisional identification thus obtained could finally be established for all compounds under study by standard methods for chemical analysis.     

Determination of the configuration of histrelin,and LHRH analog,by chiral capillary gas chromatography

Summary Summary The assigned chirality at each center of the synthetic nonapeptide histrelin (L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-N^im-benzyl-histidyl-L-leucyl-L-arginyl-L-proline-ethylamide) was verified using chiral gas chromatography. The procedure involved acid hydrolysis of histrelin to the constituent amino acids, derivatization as the N-pentafluoropropionyl/isopropyl esters and the analysis of the mixture using a commercially available 25m chiral capillary column (Chirasil-L-Val). There was no significant difference in the retention time of the amino acids obtained from the hydrolysate mixture when compared to the appropriate standards. Additionally, the hydrolysate was spiked with the D and L amino acids to prove the identity of closely eluting peaks. Luteinizing hormone-releasing hormone     

溶剂效应和取代基效应对2-(2-氨基苯基)苯并噻唑光谱性质及激发态分子内质子转移的影响

合成了多种2-(2-氨基苯基)苯并噻唑(APBT)氨基氢原子被供电子及吸电子基团取代的衍生物, 并用紫外光谱﹑荧光光谱等方法和密度泛函理论(DFT)计算研究了溶剂效应和取代基效应对衍生物的光谱性质及激发态分子内质子转移(ESIPT)的影响规律. 结果表明, 相比于非极性溶剂环己烷, 随溶剂极性的增加及APBT-溶剂分子间氢键的形成, APBT的紫外-可见最大吸收峰和荧光最大发射峰均发生了一定程度的红移, 并对APBT的ESIPT产生了影响. 在APBT分子的氨基氮原子上引入不同的吸电子或斥电子取代基, 对氮原子的电荷性质有较大的影响. 在环己烷溶剂中, 甲基取代后的APBT仅有单重荧光发射峰, 体系未发生ESIPT过程; 而COCH₂Cl等吸电子基团能促进APBT的ESIPT, 其荧光发射光谱出现了明显的双重峰, 表明体系发生了激发态分子内质子转移反应. 量子化学的理论计算较好地验证了光谱实验结果.     

Addition of Nucleophiles to Fragment Ions in Mass Spectrometers under Electrospray-Ionization Conditions

Mass spectra of several aromatic compounds containing an amino or thiol group were recorded under electrospray conditions with a Finnigan TSQ 700, as well as on a Bruker Esquire-LC spectrometer. The MS/MS spectra displayed two main peaks: the first one arising from the cleavage of the amino or thiol group; the second one appeared at 18 Da higher than the first one. It could be shown that the second peak resulted from a nucleophilic addition of one water molecule to the fragment ion.     

Identification of plant and animal glues in museum objects by GC-MS, after catalytic hydrolysis of the proteins by the use of a cation exchanger, with simultaneous separation from the carbohydrates

A method is described which enables the group-separation of proteinaceous binding media from vegetable glues (carbohydrates), and simultaneous hydrolysis of the proteins in mixtures of both. The mixtures of the binders are suspended in aqueous-ethanolic solvent with the H+ form of a strong cation exchanger and treated at elevated temperature in sealed vials. The polypeptides are cleaved by H+-catalysed hydrolysis. On abstraction the amino acids are transformed into the ammonium ions by the protons, and the cations are adsorbed by the exchanger resin. The amino acids are removed from solution in this way, thus suppressing interfering reactions with other binders, e.g. humin formation with carbohydrates. Clear and colourless solutions were obtained with all mixtures of vegetable and animal glues. Two fractions can be obtained after separation of the solid resin from the liquid supernatant - the resin fraction with the adsorbed amino acids, and the aqueous-ethanolic solution with the carbohydrates. In each of these fractions the two classes of binder can be identified separately by GC-MS; this avoids the occurrence of unresolved GC peaks and superimposed mass spectra. The method has been used to identify the binder found between fabric layers of a Burgundian liturgical vestment of the Order of the Golden Fleece from the first half of the 15th century, the Cope of the Virgin Mary. With the aid of the GC pattern obtained, and the mass spectra of the main peaks, which were identified as glucopyranose anomers, the binding medium was identified as starch.     

20种α-氨基酸的太赫兹光谱及其分子结构的相关性

应用太赫兹时域光谱(THz-TDS)技术, 在室温下对构成蛋白质的20种基本氨基酸的多晶粉末压片样品进行了光谱测试分析. 结果表明, 所有氨基酸对THz波反应非常灵敏, 在0.2-3.0 THz的有效频谱范围内, 表现出各自特征吸收峰, 故而利用THz光谱可以有效地区别不同种类的氨基酸. 我们以新数据验证和补充了前人的研究结果, 建立了以氨基酸分子结构及其THz光谱特征为基础的分类方案, 讨论并揭示了氨基酸分子的结构差异与其THz吸收光谱之间的相关性. 认知这些相关性将有助于鉴定氨基酸分子, 促进THz光谱学的理论研究以及在生物医学领域的推广应用.     

Preparation and characterization of mixed-mode monolithic silica column for capillary electrochromatography

A silica-based monolithic stationary phase with mixed-mode of reversed phase (RP) and weak anion-exchange (WAX) for capillary electrochromatography (CEC) has been prepared. The mixed-mode monolithic silica column was prepared using the sol–gel technique and followed by a post-modification with hexadecyltrimethoxysilane (HDTMS) and aminopropyltrimethoxysilane (APTMS). The amino groups on the surface of the stationary phase were used to generate a substantial anodic EOF as well as to provide electrostatic interaction sites for charged compounds at low pH. A cathodic EOF was observed at pH above 7.3 due to the full ionization of residual silanol groups and the suppression in the ionization of amino groups. A variety of analytes were used to evaluate the electrochromatographic characterization and column performance. The monolithic stationary phase exhibited RP chromatographic behavior toward neutral solutes. The model anionic solutes were separated by the mixed-mode mechanism, which comprised RP interaction, WAX, and electrophoresis. Symmetrical peaks can be obtained for basic solutes because positively charged amino groups can effectively minimize the adsorption of positively charged analytes to the stationary phase.     

Determination of amino acids in tobacco samples by capillary electrophoresis/indirect absorbance detection with isolation of the electrolysis compartment and p-Aminobenzoic acid as a background electrolyte.

A fast, convenient and sensitive method of capillary zone electrophoresis (CZE) and indirect UV detection was proposed for the determination of 16 amino acids. p-Aminobenzoic acid (PAB) was selected as a background electrolyte (BGE). An isolated cell included a BGE buffer part and an electrode buffer one, which were jointed with a glass frit. The isolated cell can prevent PAB from the electrode reaction and improve the stability of the detection baseline. The separation conditions of amino acids were investigated, such as different BGEs, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers. Under the selected separation conditions, 14 amino acid peaks could be separated in 12 min. The detection limits of the amino acids were in the range of 1.7 - 4.5 micromol/L. The isolated cell is suitable for reagents reacting on the electrodes in capillary electrophoresis. The proposed method has been successfully applied to the determination of the amino acids in tobacco samples.