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1.
V Stocchi G Piccoli M Magnani F Palma B Biagiarelli L Cucchiarini 《Analytical biochemistry》1989,178(1):107-117
A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
2.
The investigation of the stabilities of o-phthalaldehyde/2-mercaptoethanol derivatives of amino acids using a precolumn reaction technique and a high-pressure liquid chromatographic procedure is reported. The amino acid derivatives are shown to be stable on the high-pressure liquid chromatography column. Optimal conditions for the development of these derivatives for their separation using this technique are recommended. 相似文献
3.
An improved and very simple procedure for thiazolinone conversion to thiohydantoin derivatives and their separation by reverse-phase high-pressure liquid chromatography is described. Trifluoroacetic acid (10%) in ethyl acetate has been employed as a conversion reagent to circumvent the deamidation of acid amides and methylation of acidic amino acids, with a concomitant increase in the detection limits of these residues. Additionally, a very simple procedure has been developed for the separation of phenylthiohydantoin (PTH) derivatives of amino acids. The system takes advantage of the computer-controlled precise mixing of the solvents A and B to achieve accurate pH and thus avoid the necessity of pH adjustment of a buffer. The procedure is simple and highly reproducible, and separates all the 20 known PTH amino acids. The efficiency of the method has been examined on synthetic and natural proteins/peptides, in manual and autoconversion systems, over a period of more than 18 months. 相似文献
4.
R. Knecht U. Seemüller M. Liersch H. Fritz D.G. Braun J.Y. Chang 《Analytical biochemistry》1983,130(1):65-71
The complete amino acid sequence of a proteinase inhibitor, eglin c (Mr 8100), has been determined with less than 150 μg of the protein using the following microtechniques: (a) amino acid analysis with a low-nanogram amount of protein hydrolysate using dimethylaminoazobenzene sulfonyl chloride, (b) peptide isolation at the picomole level using the dimethylaminoazobenzene isothyiocyanate (DABITC) precolumn derivatization method, and (c) automatic Edman degradation. One amino acid residue has been corrected for the previously reported sequence. The Contribution of each technique to the microsequencing is discussed. In addition, a new high-performance liquid chromatography system that gives a complete baseline separation of all phenylthiohydantoin-amino acids is described. 相似文献
5.
A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl-N(tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140 degrees C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals. 相似文献
6.
Vytas-J.K. Švedas Igor J. Galaev Ivan L. Borisov Ilya V. Berezin 《Analytical biochemistry》1980,101(1):188-195
A detailed study has been made of the kinetics of interaction between amino acids and esters of amino acids and o-phthaldialdehyde in the presence of mercaptoethanol. The reaction products have been characterized. A spectrophotometric method for quantitative analysis of all amino acids, except proline and hydroxyproline, has been developed. The possibility of determination of amino acid esters in mixtures containing free amino acids has been demonstrated. It is noted that determination of glycine and histidine with the help of o-phthaldialdehyde has certain specificities associated with faster, compared to other amino acids, degradation of their derivatives. Optimal conditions for quantitative analysis of amino acids in solutions of higher than 10?5m concentration are recommended. The reproducibility of the determination was ±2%. 相似文献
7.
Kantharaju Basanagoud S. Patil Vommina V. Suresh Babu 《Letters in Peptide Science》2002,9(4-5):227-229
The chloride derivatives of all common Fmoc-amino acids lacking polar side chains as well as a number of benzyl based side chain protection have been prepared using the corresponding amino acid and thionyl chloride assisted by ultrasound has been described. The protocol is simple, efficient and rapid. All the acid chlorides prepared have been obtained in good yield and purity. 相似文献
8.
Kantharaju Patil Basanagoud S. Suresh Babu Vommina V. 《International journal of peptide research and therapeutics》2002,9(4-5):227-229
Summary The chloride derivatives of all common Fmoc-amino acids lacking polar side chains as well as a number of benzyl based side
chain protection have been prepared using the corresponding amino acid and thionyl chloride assisted by ultrasound has been
described. The protocol is simple, efficient and rapid. All the acid chlorides prepared have been obtained in good yield and
purity. 相似文献
9.
2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage 总被引:3,自引:0,他引:3
K Barlos O Chatzi D Gatos G Stavropoulos 《International journal of peptide and protein research》1991,37(6):513-520
The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp, Met, and Tyr. 相似文献
10.
Quantitative determination of phenyl isothiocyanate-derivatized amino sugars and amino sugar alcohols by high-performance liquid chromatography 总被引:4,自引:0,他引:4
Simple and rapid methods for the preparation of phenylthiocarbamyl (PTC) derivatives of amino sugars and amino sugar alcohols and their quantitative determination with high sensitivity (less than 10 pmol) by C18 reversed-phase high-performance liquid chromatography are described. Rapid sample preparation of the phenyl isothiocyanate (PITC)-derivatized amino sugars and amino sugar alcohols was achieved by a simple extraction of the reaction mixture with chloroform to remove the excess PITC and its adducts. Baseline separation of the PTC derivatives of amino sugars and amino sugar alcohols was obtained within 30 min, using a simple solvent system consisting of 0.2% each of n-butylamine, phosphoric acid, and tetrahydrofuran. The mobile phase containing n-butylamine, in conjunction with a C18 stationary phase, mimics the conditions for the separation of carbohydrates on an amino-bonded column. GlcNH2 and GalNH2 derived from the initial protein-sugar linkages were also separated from the amino acids for quantitative estimation of sugar chains in glycoproteins. Amino sugar alcohols gave single reaction products with PITC while the reaction with amino sugars was accompanied by the formation of secondary products. Apparently the secondary products were formed in an acid-catalyzed intramolecular cyclization of the PTC-hexosamines involving the aldehyde functional group. Conditions were developed to stop the transformations and maintain the stability of PTC derivatives for their convenient determination by HPLC. 相似文献
11.
Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate 总被引:81,自引:0,他引:81
Methods for the quantitative derivatization of amino acids with phenylisothiocyanate and for the separation and quantitation of the resulting phenylthiocarbamyl derivatives by reverse-phase high-performance liquid chromatography are described. Phenylthiocarbamylation of amino acids proceeds smoothly in 5 to 10 min at room temperature. Coupling solvents, reagent, and some byproducts are removed by rotary evaporation under high vacuum, and the phenylthiocarbamyl derivatives are dissolved in 0.05 M ammonium acetate, pH 6.8, for injection onto the octyl or octadecylsilyl reverse-phase column. Columns are equilibrated with the same solvent and the effluent stream is monitored continuously at 254 nm for detection of the amino acid derivatives. Elution of all of the phenylthiocarbamyl amino acids is achieved in about 30 min utilizing gradients of increasing concentrations of ammonium acetate and acetonitrile or methanol. This approach to amino acid analysis offers select advantages, both with respect to methods which employ reverse-phase separation of prederivatized samples and to the classical ion-exchange procedure. All amino acids, including proline, are converted quantitatively to phenylthiocarbamyl compounds and these are stable enough to eliminate any need for in-line derivatization. Furthermore, results comparable in sensitivity and precision to those obtained by state-of-the-art ion-exchange analyzers may be generated with equipment that need not be dedicated to a single application. 相似文献
12.
Shah AJ Crespi F Heidbreder C 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,781(1-2):151-163
Amino acids in the central nervous system can be divided into non-neurotransmitter or neurotransmitter depending on their function. The measurement of these small molecules in brain tissue and extracellular fluid has been used to develop effective treatment strategies for neuropsychiatric and neurodegenerative diseases and for the diagnosis of such pathologies. Here we describe the separation and detection techniques that have been used for the measurement of amino acids at trace levels in brain tissue and dialysates. An overview of the function of amino acid transmitters in the brain is given. In addition, the type of sampling techniques that are used for the determination of amino acid levels in the brain is described. 相似文献
13.
Abstract: A mass fragmentographic method for the determination of trace amounts of amino acid neurotransmitter candidates from brain perfusates is described. The analytical procedure includes the measurements of glycine, β-alanine, γ-aminobutyric acid, proline, aspartic acid, and glutamic acid; αalanine, leucine, and sarcosine, undergoing gas chromatographic coelution, are detected simultaneously. Amino acids extracted from dried perfusate residues are converted to the corresponding N -pentafluoropropionyl hexafluoroisopropyl esters by a single-step procedure. Gas chromatographic separation of the amino acid derivatives is achieved on a packed glass column filled with trifluoropropylsilicone as stationary phase. The limit of detection for the different derivatives (signal-to-noise, 3:1) ranges from 50 femtomol to 1 picomol. Deuterium-labeled amino acid analogues are used as internal standards for quantitative measurements. The mass spectral characteristics of the derivatives are compared and discussed. The technique has been applied to the assay of amino acids released in vivo within the pigeon optic tectum, demonstrating the capabilities of the present analytical approach. 相似文献
14.
A reverse-phase high-performance liquid chromatography procedurehas been developed for rapid separation and quantitation offree amino acids as o-phthaldialdehyde derivatives. A two stepisocratic solvent system was used which enabled an accurateanalysis at nanomole level. However, two major disadvantagesto this procedure were the lack of reaction of proline and theco-elutions of threonine/glycine and tryptophan/methionine.The free amino acids in Zea mays roots were separated by usingthe method described. Amino acids, liquid chromatography, o-phthaldialdehyde derivatives, reverse-phase chromatography, Zea mays L, maize, corn 相似文献
15.
Precolumn formation of o-phthalaldehyde (OPA) derivatives of amino acids, followed by reverse-phase separation and fluorescent detection, provides rapid, sensitive amino acid analysis. Eighteen OPA-amino acid derivatives are resolved on a Micropack MCH 5 column and can be measured at picomole levels. Ease of derivative preparation and separation makes liquid chromatographic analysis of OPA-amino acids a convenient and improved technique for measuring or confirming the presence of low levels of amino acids in aqueous solutions. Use of the method was demonstrated by measuring low concentrations of amino acids released from zooplankters stressed by contaminants. 相似文献
16.
A reliable high-performance liquid chromatography (HPLC) method is described for the separation of dimethylamino-azobenzenesulfonyl-amino acid (DABS-AA). The separation is accomplished by reversed-phase chromatography on a Hypersil ODS column (4.6×150 mm) withe a Hewlett-Packard liquid chromatography system. In addition to the developed sample and solvent preparation procedure, this precolumn modification method using dimethylaminoazobenzene sulfonyl chloride (DABS-CL) for amino acid analysis is proved reliable and sensitive. Five apolipoprotein B-100 tryptic peptides, two of them containing cysteine, were demonstrated as examples for the general application of this method in amino acid analysis. It is a useful method for analysis of cysteine- and cysteine-containing peptide and, furthermore, for determination of sulfhydryl and disulfide linkages of proteins. 相似文献
17.
Zden
k Deyl Ivan Mik
ík 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,699(1-2)
Analytical methods for marker compounds formed during post-translational modifications of proteins are reviewed. Only adducts arising either in vivo or under in vitro conditions simulating the in vivo situations are discussed. All of these compounds stem from either the reaction of free amino groups (i.e., lysine, arginine or N-terminal amino acid). In most cases the reactive counterpart is an aldehydic moiety containing endogenous compound; however, other functional groups containing metabolites are considered as well. The main demand put upon such marker compounds is that they are stable in acid or enzymatic hydrolysis or, alternatively, can be stabilized by simple sample pretreatment (e.g., by reduction). Practically all categories of separation procedures can be applied provided that the chemical characteristics of a particular marker are adequately respected; frequently combination of two different separation procedures based on different principles must be used. Because of the low level of such marker compounds under in vivo conditions, an appropriate sample enrichment step must be involved. Emphasis is put upon the analysis of Amadori products, pentosidine (and pentosidine related compounds), pyrraline, furosine, N-carboxymethylamino acids, amino acid hydantoins and stabilized dicarbonyl intermediates 相似文献
18.
Summary. A fluorescent electrophilic reagent, 9-fluorenone-4-carbonyl chloride (FCC), is chosen to functionalize amino acids in alkaline
medium before their HPLC resolution. FCC reacts with both primary and secondary amino acids to produce stable and highly fluorescent
derivatives suitable for sensitive and efficient chromatographic determination and resolution on a teicoplanin chiral stationary
phase (CSP) using the methanol-based solvent mixture as the mobile phase. The detection limit is in the picomole range and
approximately 0.01% of the d-enantiomer in an excess of the l-enantiomer is detectable. However, the resolution is not reproducible under the elution of either the water- or the acetonitrile-based
mobile phase. The increase in solubility of analyte in the mobile phase seems to be responsible. Upon comparison under the
optimal chromatographic conditions, the resolution is better than that for the 9-fluorenylmethyl chloroformate (FMOC) or 6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate (AQC) derivatives reported previously. 相似文献
19.
A method is described for the determination of γ-amino butyric acid, glycine, glutamine, glutamate, asparate, and taurine in small samples of brain tissue. This is an extension of a method based on the formation of derivatives with [3H]dansyl chloride (15) with the addition of 14C-labeled amino acids as internal standards to allow correction for incomplete and variable degrees of dansylation. An improved chromatographic separation of dansyl-taurine is employed. Estimates of the contents of these amino acids in rat cortical tissue are reported and compared with those obtained on an autoanalyser, and with those in the literature using autoanalytical and other methods. 相似文献
20.
4'-Dimethylaminoazobenzene-4-sulfonyl chloride is a chromophoric reagent commonly used to detect amino acids at picomole levels. This article describes a single-column reverse-phase high-performance liquid chromatography system which allows the resolution and analysis of the dabsyl chloride derivatives of several modified amino acids. Highly derivatized C18 (22 and 31%) columns from Phenomenex are run at pH 8.1 to separate dityrosine, the phosphorylated amino acids (o-phosphoserine, o-phosphothreonine, and o-phosphotyrosine), and the 17 other amino acids normally present in protein hydrolysates. In order to gain additional sensitivity and to verify the presence of dityrosine, the dityrosine and lysine peaks are collected and run at pH 4.1 on the same columns. Our experience indicates that, with the described setup, the lower limit for accurate and reproducible detection is near 1 pmol. This method has been applied to the analysis of dityrosine in uv-irradiated calmodulin and cardiac troponin C and to the detection of phosphorylation sites in several polypeptides. 相似文献