Quantification of human growth hormone by amino acid composition analysis using isotope dilution liquid-chromatography tandem mass spectrometry

We describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-HPLC-mass spectrometry (ID-HPLC-MS) method. Sample purity was confirmed using capillary zone electrophoresis, HPLC and MS. The analyte protein, human growth hormone (hGH), was effectively hydrolyzed by incubation with 8 M hydrochloric acid at 130 °C for 48 h, where at least 1 μM of hGH was treated to avoid possible degradation of released amino acids during hydrolysis. Using a reversed-phase column, the analytes (isoleucine, phenylalanine, proline and valine) were separated within 5 min using an isocratic eluent comprising 10% acetonitrile containing 0.1% trifluoroacetic acid. The detection limit (signal to noise ratio of 3) of amino acids was 5.5-6.2 fmol per injection. The quantification precision (RSD) of amino acids for intra- and inter-day assays was less than 0.98% and 0.39%, respectively. Comparison with other biochemical and instrumental methods revealed substantially higher accuracy and reproducibility of the ID-HPLC-MS/MS method as expected. The optimized hydrolysis and analytical conditions in our study were suitable for accurate quantification of hGH.     

Accurate evaluation method of the polymer content in Monomethoxy(polyethylene glycol) modified proteins based on Amino acid analysis

To overcome the uncertainty of the colorimetric or fluorimetric method so far employed for the evaluation of monomethoxy(polyethylene glycol) (MPEG) covalently bound to protein, a direct method based on amino acid analysis is proposed. The method exploits the use of MPEG, which was bounded with the unnatural amino acid norleucine (MPEG-Nle). MPEG-Nle was activated at its carboxylic group to succinimidyl ester for the binding to the amino groups of protein. After acid hydrolysis, the amino acid content is evaluated by conventional amino acid analyzer or by reverse-phase HPLC as phenylthiocarbamyl derivative. The number of bound MPEG chains is calculated from the amino acid composition, since one norleucine residue is released from each bound polymer chain. The method was verified with several proteins in comparison with colorimetric ones, also in the case of proteins that contain chromophores in the visible range, such cytocrome C. It was observed that in most of the cases, the colorimetric methods give an overestimation of the degree of protein modification.     

Studies on pyrazines. 4.. The synthesis of 2-hydroxy-6-phenylpyrazine and its derivatives

This report describes a new method for the preparation of 2-hydroxy-6-phenylpyrazine ( 1 ). Amino acetal 5 was converted to glycyl amino acetal 7 by two steps in excellent yield. Cycliza-tion of 7 to 1 was accomplished in 33% yield by refluxing in acetic acid followed by oxidation with manganese dioxide. Compound 1 was also prepared by hydrolysis of amino- and methoxy-pyrazines 3 and 15 , derived from 2-hydroxy-5-phenylpyrazine ( 2 ) and the 2-amino homologue 4 , respectively, and by decarboxylation of 2-hydroxy-5-phenylpyrazinecarboxylic acid ( 19 ).     

The effect of hydrolysis time on amino acid analysis

Determining the amino acid content of a protein involves the hydrolysis of that protein, usually in acid, until the protein-bound amino acids are released and made available for detection. Both the variability in the ease of peptide bond cleavage and differences in the acid stability of certain amino acids can significantly affect determination of a protein's amino acid content. By using multiple hydrolysis intervals, a greater degree of accuracy can be obtained in amino acid analysis. Correction factors derived by linear extrapolation of serial hydrolysis data are currently used. Compartmental modeling of the simultaneous hydrolysis (yield) and degradation (decay) of amino acids by nonlinear multiple regression of serial hydrolysis data has also been validated and applied to determine the amino acid composition of various biological samples, including egg-white lysozyme, human milk protein, and hair. Implicit in the routine application of serial hydrolysis in amino acid analysis, however, is an understanding that correction factors, derived either linearly or through the more accurate nonlinear multiple regression approach, need to be determined for individual proteins rather than be applied uniformly across all protein types.     

Quantitative determination of the ligand content in Benzamidine Sepharose 4 Fast Flow media with ion-pair chromatography

A quantitative hydrochloric acid hydrolysis-HPLC method was developed for the analysis of the ligand content of Benzamidine Sepharose 4 Fast Flow media. The method requires about 100 mg of dried sample and simple reaction vials can be utilised. Release of the ligand (p-aminobenzamidine) from the base matrix (Sepharose 4 Fast Flow) was obtained after hydrolysis for 180min at 70 degrees C in concentrated hydrochloric acid. When Benzamidine Sepharose 4 Fast Flow media were treated this way p-aminobenzoic acid and p-aminobenzamidine were the only products released from the ligand. A chromatographic system based on ion-pair reversed phase separation was used to quantify these ligand products. The mobile phase was made acidic enough to make p-aminobenzoic acid and p-aminobenzamidine positively charged in order to make ion-pair formation with hexanesulfonic acid possible. The relative standard deviation of th e method was below 2% and no systematic errors could be detected when the results were compared to an independent method based on elemental analysis (nitrogen). The new HPLC method was used to analyse ligand densities in the range of 2-20 micromol/ml medium.     

Determination of the protein and free amino acid content in a sample using o-phthalaldehyde and N-acetyl-L-cysteine

A spectrophotometric method is proposed for determining the protein content in a sample after total acid hydrolysis. In the procedure, free amino acids are caused to react with o-phthalaldehyde and N-acetyl-L-cysteine at pH 9.5, using isoleucine as the reference compound. Correction factors are used to take into account the differences between the molar absorptivities of the amino acid isoindoles and the recoveries of the amino acids after the hydrolysis treatment. The limit of detection was in the range 40-50 micrograms of protein, and the recoveries were usually 101 +/- 3% with a coefficient of variation lower than 4%. The free amino acid content in a partially hydrolysed protein was also determined.     

Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography–tandem mass spectrometry

Herein, we describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography–tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 mol/L hydrochloric acid at 130 °C for 48 h, in which at least 1 μmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline, and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mmol/L ammonium acetate, both containing 0.3 % (v/v) trifluoroacetic acid. The optimized hydrolysis and analytical conditions in our study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.

Applications of gas-liquid chromatography in protein chemistry. Determination of C-terminal sequences on nanomolar amounts of proteins.

A method for the determination of C-terminal amino acids and C-terminal amino acid sequences in nanomolar amounts of proteins is described, based on carboxypeptidase A digestion of the protein, followed by removal of the partially digested protein and quantitative gas-liquid chromatographic determination of the amino acids released after known time intervals. Sequences deduced from the kinetics of release of specific amino acids are compared with the known C-terminal sequences of well-characterized proteins.     

Characterization of proteinaceous glues in old paintings by separation of the o-phtalaldehyde derivatives of their amino acids by liquid chromatography with fluorescence detection

A HPLC-fluorescence method for characterization of proteinaceous glues from binding media used in pictorial works of art prior to conservation or restoration treatment is proposed. Fluorescence derivatization of amino acids released by acid hydrolysis of standard proteins is studied. The derivatization reagent was o-phtalaldehyde with 2-mercaptoethanol as catalyst. Mobile phase was a programmed gradient among two eluents (water buffered at pH 5.8 wit 5% THF, and methanol) and is able to satisfactorily resolve the amino acid derivatives in 45 min. Peak area ratios among amino acid derivatives and the leucine derivative are useful to characterize the proteins. The method shows good sensitivity and adequate linearity between 2.0 × 10⁻³ and 3.3 mmol/l of each amino acid, with a limit of detection of 6.0 × 10⁻⁴ mmol/l. The proposed method has been successfully applied to artistic samples from items of the cultural heritage of Valencia (Spain).     

Chromatographic amino acid analysis of protein hydrolysed in polyacrylamide after removal of ammonia

Summary A simple method for the removal of NH₃ from amino acids is presented. The method is based on a cation-exchange resin from which amino acids are eluted with NH₄OH. The eluent is then removed under reduced pressure. The method allows the ninhydrin-based detection of amino acids after hydrolysis of stained protein bands in polyacrylamide gels. This was previously not possible since NH₃ produced by the hydrolysis of polyacrylamide interferes with the ninhydrin-detection of basic amino acids. The method should also be applicable to the detection of amino acids with o-phthalaldehyde.     

The environment controlled effect of thiacalix[4]arene on the transition thermodynamics and kinetics of bovine serum albumin

Complexation ability of water-soluble thiacalix[4]arene-tetrasulfonate towards three aromatic amino acids (Phenylalanine, Tyrosine and Tryptophane) was studied in water–ethanol mixtures by photoluminescence (PL) method as a function of the ethanol content of the bulk solutions. Job’s method followed by the application of the van’t Hoff theory was used to determine the thermodynamic parameters of the molecular association. Results show quite different thermodynamics of formation of calixarene–amino acid complexes at low and higher ethanol content of the solutions. The considerable stability of the individual calixarene–aromatic amino acids complexes supports their existence also in the case when the amino acids are in a protein. To test this idea the conversion rate, enthalpy and entropy change associated to the structural transition of BSA (bovine serum albumin) were investigated by Differential Scanning Calorimetry (DSC) in the absence and in the presence of calixarene. Results show that presence of calixarene changes significantly both the thermodynamics and the kinetics of the transition of BSA and the information collected for the individual calixarene–amino acid complexes gives insights about the possible processes at molecular level.     

一种新的玉米抗氧化肽的制备与结构表征

用酶法在受控条件下降解玉米醇溶蛋白,对酶解产物进行分离纯化,获得一种新的抗氧化肽;对其一级结构进行了表征,氨基酸组成和顺序为Leu-Asp-Tyr-Glu;从结构上分析了其可能的抗氧化机制.     

Methodology for determining degree of hydrolysis of proteins in Hydrolysates: a review

Degree of hydrolysis (DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate. Several methods exist for determining DH; the most commonly used of these include the pH-stat, trinitrobenzenesulfonic acid (TNBS), o-phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), and formol titration methods. The pH-stat method is based on the number of protons released during hydrolysis; the TNBS, OPA, and formol titration methods are based on the measurement of amino groups generated from hydrolysis. The SN-TCA method measures the amount of TCA-soluble nitrogen, rather than DH. The pH-stat is the simplest and most commonly used method, but does not determine peptide bonds directly. In addition, the accuracy of the method depends on the type of hydrolytic enzymes used, the size of the hydrolyzed peptides, and the reaction temperature. Generally, the TNBS and OPA methods compare well and do directly determine DH. However, the assumption that the response factor for all derivatized N-terminal amino acids is similar may lead to inaccuracies. In conclusion, there is no consensus as to the best method for determining the DH of protein hydrolysates; consequently, there is a need for a standardized approach if interstudy comparisons are to be made.     

Rapid Protein Hydrolysis by Microwave Irradiation Using Heat-Resistant Teflon-Pyrex Tubes

A rapid peptide-bond hydrolysis by means of microwave irradiation is introduced for the facile preparation of protein hydrolysates used for amino acid analysis. The optimal hydrolysis condition has been determined using several enzymes with known amino acid compositions. The effects of hydrolysis time on the recovery of various labile and hydrophobic amino acids are also exemplified in the microwave heating of standard amino acids. The method has been applied to the complete amino acid analysis with a single nonvolatile solvent of methanesulfonic acid with good recovery of tryptophan and half-cystine. It provides a radical expedition of protein and peptide hydrolysis via commercial microwave ovens and specially-designed Teflon-Pyrex tubes, circumventing the tedious procedures using vacuum-sealed pyrex lubes heating at 110°C for more than 24 h. This novel type of microwave chemistry associated with rapid peptide-bond cleavage is of great potential in the automation of the complete process of amino acid analysis starting from the preparation of protein hydrolysates.     

牛颌下腺粘蛋白中糖组成的毛细管气相色谱分析

根据各种糖革键稳定性的差异，采用没的酸解方法，使各种单糖从牛颌下腺粘蛋白上游离下来。纯化后衍生成三甲基硅醚或糖醇乙酸酯，应用毛细管气相色谱技术分析。结果表明，牛颌下腺粘蛋白中含三类糖，即唾液酸、中性糖和氨基糖。每咱单糖的含量分别为５－Ｎ－乙酰神经氨酸４．３％；５－Ｎ＝乙醇酰神经氨酸１９．０％；岩藻糖３．２％；半乳糖５．１％；Ｎ－乙酰氨基半乳糖１８．４％；Ｎ－乙酰氨基葡萄糖８．７％。     

Assignment of the configurations of the amino acids in peptidic siderophores

Pyoverdins and azotobactins contain beta-hydroxyaspartic acid, N delta-hydroxyornithine, citrulline and homoserine, in addition to the common protein amino acids. Configuration assignment of all of these was achieved by acid hydrolysis of the peptide, derivatization of the constituent amino acids to the N-pentafluoropropionyl amino acid esters and gas chromatographic separation of the stereoisomers on capillaries coated with Chirasil-Val. This approach is straightforward for the protein amino acids, but the less common amino acids are either partially degraded during acid hydrolysis or their derivatives exhibit unfavourable gas chromatographic properties. By judicious combination of partial and total hydrolysis and dual derivatization, these problems may be overcome.     

Direct infusion mass spectrometry as a fingerprint of protein-binding media used in works of art

A direct infusion mass spectrometry method for the characterization of proteinaceous glues from binding media used in pictorial works of art prior to conservation or restoration treatment is proposed. Amino acids are released by acid hydrolysis and dissolved in a mixture of acidic water and ethanol. This mixture is directly infused into a mass spectrometer without any derivatization. The mass spectrometer is operated in positive ion electrospray mode (ESI-MS) to yield [M+H](+) ions for the amino acids. Relative amounts of each amino acid are calculated for each protein (beef and porcine gelatines, albumin, casein and egg). The analyzed proteins were satisfactorily distinguished. The method is easy and fast, and shows good sensitivity and resolution. The proposed method has been successfully applied to artistic samples from items of the cultural heritage of Valencia (Spain).     

Zur Synthese sulfonierter Derivate von 4-Fluoranilin

Syntheses of Sulfonated Derivatives of 4-Fluoroaniline Synthesis of 2-amino-5-fluorobenzenesulfonic acid ( 2 ) was achieved by baking the hydrogen sulfate of 4-fluoroaniline ( 1 ). Sulfonation of p-fluoroacetanilide ( 4 ) with oleum followed by hydrolysis gave 5-amino-2-fluorobenzenesulfonic acid ( 3 ). The same reaction with 1 yielded 3 in an impure state. The structures of 2 and 3 were confirmed by converting the diazonium chlorides derived from 5-fluoro-2-nitroaniline ( 5 ) and from 2-fluofo-5-nitroaniline ( 8 ) to 5-fluoro-2-nitrobenzene-sulfonyl chloride ( 6 ) and 2-fluoro-5-nitrobenzenesulfonyl chloride ( 9 ), respectively, followed by hydrolysis of 6 to 5-fluoro-2-nitrobenzenesulfonic acid ( 7 ), and of 9 to 2-fluoro-5-nitrobenzenesulfonic acid ( 10 ), and by final reduction. Compound 10 was also obtained by sulfonation of 1-fluoro-4-nitrobenzene ( 11 ) with oleum.     

气相色谱-燃烧-同位素比值质谱法分析氨基酸氮稳定同位素并初步评估水生生物体营养级

氨基酸稳定氮同位素(δ15 N)分析能准确有效地评估生物体的营养级以及氮在食物链中的流动.本研究优化了氨基酸氮同位素的分析方法:样品在酸性条件下水解后,释放出的蛋白质氨基酸经阳离子交换树脂纯化后,衍生为对应的N-新戊酞基,O-异丙醇(N-pivaloyl-isopropyl,NPP)酯,利用气相色谱-燃烧-同位素比值质谱仪(Gas chromatography-combustion-isotope ratio mass spectrometry,GC-C-IRMS)测定其δ15 N.经非极性气相色谱柱DB-5ms分离后,13种氨基酸NPP酯衍生物均可得到良好的基线分离.在样品量不低于20 ng N条件下,GC-C-IRMS方法的精密度优于1‰,测得的δ15 N值与EA-IRMS法测得的δ15 N值没有明显差异.阳离子树脂纯化前后各氨基酸δ15 N值差异低于1‰,表明没有产生明显的同位素分馏.采用本方法成功地估算了阿哈湖生态系统中常见水生生物的营养级,可作为研究氨基酸代谢以及生态系统特征的新方法.     

Sphingosine determination at the picomole level using dimethylaminoazobenzene sulphonyl chloride

A sensitive method (lower detection limit 5 picomoles) is proposed for the determination of sphingoid bases liberated from mammalian glycosphingolipids by acid hydrolysis. The azo dye 4-dimethylaminoazobenzene-4'-sulfonyl chloride reacts with the primary amino group of the sphingosine bases, forming a stable derivative. Excess reagent, which is degraded during the derivatization, and free amino sugars as common hydrolysis products of glycosphingolipids are completely separated by reversed-phase high-performance liquid chromatography. This method was applied to the determination of the glycosphingolipid content of mouse spleen and thymus.