Metastable transitions in the mass spectra of methyl and phenylthiohydantoin derivatives of amino acids

The mass spectra of a number of methyl- and phenylthiohydantoin amino acid derivatives have been obtained. The major metastable transitions occurring in the mass spectra of these derivatives have been identified and measured. The major fragmentation pathways associated with the metastable transitions have been outlined and are discussed for each group of compounds. Inspection of the metastable data has shown that there is at least one unique metastable transition occurring for each thiohydantoin derivative which may be used to uniquely identify that derivative in the presence of a mixture of thiohydantoin derivatives obtained from the Edman degradation of a peptide or protein. The use of metastable ions to uniquely identify thiohydantoin derivatives in mixtures has proven useful in the identification of the MTH and PTH derivatives of glycine whose molecular ions are not unique and for resolving such ambiguities as occur for example in the mixture of leucine and isoleucine.     

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.     

Improved TLC systems for rapid resolution of phenyl thiohydantoin amino acids

Two new solvent systems, n-hexane + propionic acid (26:5, v/v) and chloroform + acetone (29:3, v/v), for the rapid resolution and identification of an 18-component mixture of phenylthiohydantoin amino acids are reported. Using these systems certain difficult combinations of phenylthiohydantoin amino acids are resolved. Two more solvent systems, viz chloroform + acetic acid (27:3, v/v) and chloroform + methanol (30:4, v/v), are developed to resolve phenylthiodantoin derivatives of aspartic and glutamic acids.     

Miniaturized protein microsequencer with PTH amino acid identification by capillary electrophoresis I. An argon pressurized delivery system for adsorptive and covalent sequencing

The design of a simple, highly miniaturized instrument for manual microsequence analysis of proteins and peptides is described. The reaction chamber is made of fused silica capillary tubing with all reagents and solvents necessary for coupling and cleavage delivered via two valves and a syringe-based dispenser. Only two pressure regulators are required. A section of the flow-through reaction chamber is heated by thermoelectric modules to control the sequencing reaction temperature. Conversion of the extracted amino acid product to the more stable phenylthiohydantoin (PTH) form is performed off-line so that it may be dissolved in 1 mu1 buffer for identification. Approximately 0.1% of this PTH product is analyzed by micellar electrokinetic capillary chromatography (MECC) with thermo-optical absorbance detection (TOAD), providing femtomole detection of the phenylthiohydantoin amino acids. Preliminary results at the 50 pmol level for both adsorptive and covalent sequencing methods are presented.     

Chromatographic Analysis of Suberimidate-Crosslinked Lysine

Abstract

Quantitative analysis of bislysylsuberamidine and monolysylsuberamidinic acid, which are obtained by an acid hydrolysis of protein cross-linked with dimethyl suberimidate, on an amino acid analyzer are described. Both of ninhydrin and fluorometric detection with o-phthalaldehyde were applied and less than 50 pmol of cross-linked lysine was analyzed in the latter case. The first-order rate constant for hydrolysis of amidine bond under standard conditions of acid hydrolysis of protein was found to be 3.4 × 10^?3h^?1.     

Rapid Acid Hydrolysis of Albumin in a Microwave Oven

Microwave-oven-assisted acid hydrolysis of albumin in 7.5 M HCl was carried out in only 12 min in a pressurized Teflon reactor using a power of 390 W. The effect of experimental conditions on the hydrolysis of albumin were evaluated and the results obtained were compared with those found by conventional hydrolysis, using in both cases the ninhydrin reaction for the spectrophotometric determination of free amino acids. A chromatographic study showed that microwave oven heating did not change the nature of the amino acids obtained from the hydrolysis of the protein.     

Fast separation of amino acid phenylthiohydantion derivatives by HPLC on a non-porous stationary phase

Summary A new method is described for the separation of phenylthiohydantoin (PTH) derivatives of the 20 common amino acids. The analysis requires approximately 7 minutes and good resolution is obtained by RP-HPLC on columns packed with a non-porous stationary phase (Kovasil-C14; 33×4.6 mm). Gradient elution was chosen with eluents containing either sodium acetate/ acetic acid buffers (moderately acidic conditions) or a heptafluorobutyric acid modifier (strongly acidic eluent). A slightly different elution order of the PTH-amino acids was found in the two systems. Low detection limits (in the femtomol range) were achieved with simple commercial HPLC equipment. Presented at the Balaton Symposium on High Peformance Separtion Methods, Siófok, Hungary, september 1–3, 1999     

Rapid analysis of ricin using hot acid digestion and MALDI‐TOF mass spectrometry

Ricin is a protein toxin of considerable interest in forensics. A novel strategy is reported here for rapid detection of ricin based on microwave‐assisted hot acid digestion and matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometry. Ricin samples are subjected to aspartate‐selective hydrolysis, and biomarker peptide products are characterized by mass spectrometry. Spectra are obtained using post source decay and searched against a protein database. Several advantages are offered by chemical hydrolysis, relative to enzymatic hydrolysis, notably speed, robustness, and the production of heavier biomarkers. Agglutinin contamination is reliably recognized, as is the disulfide bond strongly characteristic of ricin.     

Preparation of chiral stationary phase via activated carbamate intermediate for liquid chromatographic optical resolution

Summary Chiral stationary phases (CSPs) for liquid chromatography were prepared by the way of an activated carbamate intermediate. The amino group of aminopropylsilyl silica gel was first activated by carbamylation with disuccinimido carbonate (DSC). The obtained activated carbamate silica gel (ACsil) proved useful as an intermediate for the preparation of urea-type CSPs. The reaction of ACsil with (S)- of (R)-1-(α-naphthyl)-ethylamine gave naphthylethylurea type CSPs. These CSPs were also obtained directly from aminopropylsilyl silica gel by its reaction with optically active (S)- or (R)-succinimido 1-(α-naphthyl)ethyl carbamate (SINEC). Several phenylthiohydantoin amino acid enantiomers and p-bromophenylcarbamyl amino acid enantiomers were resolved on the CSPs by elution with aqueous mobile phase.     

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein.     

Methylthiohydantoin amino acids: chromatographic separation and comparison to phenylthiohydantoin amino acids

Most phenylthiohydantoin (PTH) amino acids and most methylthiohydantoin (MTH) amino acids may be separated from one another by thin-layer chromatography (TLC) using the same sequential development technique with the same two solvents. Similarly, a single solvent system may be used in high-performance liquid chromatography (HPLC) to separate most PTH-amino acids and most MTH-amino acids. When both TLC and HPLC separations are performed on a sample, all MTH-and PTH-amino acids can be uniquely identified. Since many solid-phase protein sequencing techniques generate both MTH-and PTH-amino acids, these analytical systems simplify identification of the amino acid derivatives. Although the chromatographic properties of MTH-and PTH-amino acids are similar, they are not identical (contrary to a previous report).     

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.     

Studies on Different Hydrolysis Procedures for Drugs of Abuse in Hair

1.IntroductionDrugsofabusehavebecomeamajorprobleminoursocietyinrecentyearsandhaveresultedinwidespreadabuse.Thedetectionofdrugsofabuseinbiologicalsamplesisoneoftheprimaryfunctionsofaclinicalorforensictoxicologylaboratory.Particularlyincaseswheretheres...     

Quantitative analysis of immobilized proteins and protein mixtures by amino acid analysis

Biomolecular surface engineering of materials often requires precise, versatile and efficient quantification of immobilized proteins at solid surfaces. Acidic hydrolysis of surface-bound proteins and subsequent HPLC analysis of fluorescence-derivatized amino acids were adapted and critically evaluated for that purpose. Contaminations and concentration-dependent amino acid retrieval during HPLC were found to influence the accuracy of the method. In addition to the choice of adequate conditions for hydrolysis, derivatization and chromatographic separation extensions of the data evaluation were suggested to improve the accuracy of the approach when applied to single protein systems: comparing the experimentally obtained amino acid ratio to the protein constitution enabled to identify the properly separated and detected amino acids. Those amino acids were selected for a more precise calculation of the amount of immobilized protein. To further increase the accuracy of the method, the retrieval of amino acids corresponding to protein amounts in the range between 0.5 and 4.0 microg was analyzed for a variety of proteins of interest to derive protein-specific correction factors. The evaluation of amino acid data was furthermore applied to quantify binary protein mixtures at similar settings. This method was proven useful to detect the composition of protein mixtures throughout a wide range of absolute and relative concentrations.     

Capillary electrochromatography with thermo-optical absorbance detection for the analysis of phenylthiohydantoin-amino acids.

Capillary columns were packed with 3 microns C18 stationary phase, interfaced with an ultraviolet-laser based thermo-optical absorbance detector, and evaluated for separation of a mixture of phenylthiohydantoin-amino acids. These columns demonstrated consistent performance with a relative standard deviation (RSD) for migration time of less than 1.5% and a separation efficiency of 216,000 plates/m for the electroosmotic flow marker, thiourea. The thermo-optical absorbance detector was based on a 248 nm krypton-fluoride excimer laser. Detection limits (3 sigma) ranged from 1.6 to 4.8 x 10(-7) M phenylthiohydantoin (PTH)-amino acid injected onto the column, which is a factor of three superior to those obtained in micellar electrokinetic chromatographic analysis of these compounds. A mixture of 17 PTH amino acids was injected onto the capillary; 13 components were nearly baseline resolved in 14 min.     

Strategies for internal amino acid sequence analysis of proteins separated by polyacrylamide gel electrophoresis

An evaluation has been made of various strategies for obtaining internal amino acid sequence data from electrophoretically separated proteins. Electroblotting, in situ proteolysis and extraction, and direct electroelution are compared. Electroblotting of protein or peptides from gels resulted in poor yields (typically, 1-7%). However, higher yields (3-67%) were achieved by in situ enzymatic cleavage followed by acid extraction of the peptides from the gel. Peptides extracted from the gel were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), on short, small-bore columns (100 x 2.1 mm I.D.), to enable recovery of peptides in small volumes (ca. 50 microliters) suitable for microsequence analysis. Capillary zone electrophoresis under acidic conditions (pH 2.5) was used to assess peptide purity before sequence analysis. Cysteine residues were identified in unmodified proteins or peptides by a characteristic phenylthiohydantoin (PTH)-amino acid derivative during sequence analysis. This derivative does not co-chromatograph with any known PTH-amino acid. Direct electrophoretic elution of protein from gels yielded between 45-50% of applied protein. Proteins recovered from gels by electrophoretic elution required further purification by inverse-gradient RP-HPLC [R. J. Simpson, R. L. Moritz, E. C. Nice and B. Grego, Eur. J. Biochem., 165 (1987) 21] to remove sodium dodecylsulphate and acrylamide-related contaminants for sequence analysis.     

Selenomethionine Extraction from Selenized Yeast: an LC-MS Study of the Acid Hydrolysis of a Synthetic Selenopeptide

A synthetically prepared seleno-peptide (AHPDVLTVXLQMLDDGR) was used as a model system for the acid hydrolysis of selenized yeast proteins. The seleno-peptide is a tryptic peptide of a heat shock protein 104 from Saccharomyces cerevisiae, was subjected to acid hydrolysis using methanesulfonic acid over a time period of 8 hours. Aliquots of the solution were sub-sampled at predetermined time intervals and the peptide fragments characterized by reversed phase LC MSⁿ. Similarly, the appearance of amino acid residues in the solution was monitored. It was found that after about 8 hours the synthetic peptide completely hydrolyzed. The use of a selenopeptide as a model for hydrolysis of selenized yeast hydrolysis was validated by comparing the decomposition time profile of the synthetic peptide with that of a selenized yeast sample. The rate of hydrolysis was identical in both systems, suggesting that the employed acid hydrolysis yields to the complete decomposition of the Se containing proteins in yeast and consequently to the liberation of selenomethionine.     

Determination of amino acids by capillary electrophoresis-electrospray ionization-mass spectrometry: an evaluation of different protein hydrolysis procedures

In this work, a CE equipment, online hyphenated to an IT MS analyzer by a linear sheath liquid interface promoting ESI, was used to develop a method for quantitative determination of amino acids. Under appropriate conditions (BGE composition, 0.8% HCOOH, 20% CH3OH; sheath liquid composition, 0.8% HCOOH, 60% methanol; V ESI, +4.50 kV), analytical curves of all amino acids from 3 to 80 mg/L were recorded presenting acceptable linearity (r >0.99). LODs in the range of 16-172 micromol/L were obtained. BSA, a model protein, was submitted to different hydrolysis procedures (classical acid and basic, and catalyzed by the H+ form of a cation exchanger resin) and its amino acid profiles determined. In general, the resin-mediated hydrolysis yields were overall similar or better than those obtained by classical acid or basic hydrolysis. The resulting experimental-to-theoretical BSA concentration ratios served as correction factors for the quantitation of amino acids in Brazil nut resin generated hydrolysates.     

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.     

蛋白质水解阶段对氨基酸组成分析的影响

水解是蛋白质氨基酸组成分析的重要步骤，水解质量的好坏直接影响到分析结果的正确与否，蛋白质的水解受到很多因素的影响，如温度，时间、水解试剂，添加剂，水解方法等，本文对近10年来蛋白质组成分析中中有关蛋白质水解问题的研究进进行了评述，对最近几年出现的一些新的水解方法如酸性水解中的气相水解，微波辐射水解，膜上蛋白质和印迹水解，聚丙烯酰安凝胶中蛋白质的水解，敏感氨基酸的水解以及水解过程中影响氨基酸外消旋化的因素等作了简要的介绍。