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PubMed1996

Optical resolution of phenylthiohydantoin-amino acids by capillary electrophoresis and identification of the phenylthiohydantoin-D-amino acid residue of [D-Ala2]-methionine enkephalin.

This is an initial report to propose a protein sequence analysis system with DL differentiation using capillary electrophoresis (CE). This system consists of a protein sequencer and a CE system. After fractionation of phenyl-thiohydantoin (PTH)-amino acids using a protein sequencer, optical resolution for each PTH-amino acid is performed by CE using some chiral selectors such as digitonin, beta-escin and others. As a model peptide, [D-Ala2]-methionine enkephalin (L-Tyr-D-Ala-Gly-L-Phe-L-Met), was used and the sequence with DL differentiation was determined, with the exception of the fourth amino acid, L-Phe, using our proposed system.

Amino Acids↗

PubMed1989

Protein modification by diazotized arsanilic acid: synthesis and characterization of the phenylthiohydantoin derivatives of azobenzene arsonate-coupled tyrosine, histidine, and lysine residues and their sequential allotment in labeled peptides.

Analytical procedures are elaborated for the sequential allotment of azobenzene arsonate binding sites in proteins and peptides. The reaction of diazotized arsanilic acid with proteins leads to covalent modification of tyrosine, histidine and, in part, lysine residues. Synthetic peptides containing these amino acids were modified with diazotized arsanilic acid and subjected to N-terminal sequence analysis. The amino acid derivatives phenylthiohydantoin(Pth)-azobenzene-arsonate-tyrosine, Pth-azobenzene-arsonate-histidine, and alpha-Pth-epsilon-hydroxycaproic acid are recovered upon Edman degradation of selected peptides. Phenylthiohydantoins of modified and nonmodified amino acids are fully separated by reverse-phase HPLC on a Zorbax-PTH column. For identification purposes, phenylthiohydantoins of azobenzene arsonate-labeled amino acids have been synthetized. They are characterized with respect to spectral absorption characteristics and retention times on reverse-phase supports.

Amino Acid Sequence↗

PubMed1984

Identification of amino acid phenylthiohydantoins by multicomponent analysis of ultraviolet spectra.

Mixtures of amino acid phenylthiohydantoins ( PTHs ) can be identified and quantitated by multicomponent analysis of their ultraviolet spectra. For this analysis, the direct absorbance spectra were converted to their first derivatives. Then the multicomponent analysis routine resident in the spectrophotometer was used. The entire process requires less than one minute. Mixtures of two amino acid phenylthiohydantoins with extreme spectral similarity were accurately quantitated with the method. (We used mixtures of PTH-Asp-PTH-Glu and PTH-Ile-PTH-Leu.) The ability to identify amino acid phenylthiohydantoins by analysis of their ultraviolet spectra may provide a simple method for confirmation of the identification of the amino acid PTH obtained during protein sequencing.

Amino Acid Sequence↗

PubMed1976

[Separation by high-performance liquid chromatography of phenylthiohydantoin derivatives of amino acids released by Edman degradation (author's transl)].

The separation by high-performance liquid chromatography of seventeen phenylthiohydantoin derivatives of amino acids released after Edman degradation of proteins is described. Sixteen phenylthiohydantoin derivatives of amino acids are differentiated within 40 min by liquid-liquid partition on a column packed with Micropak CN (moderately polar alkyl nitrile phase bonded on 10mum porous silica microparticles) and gradient elution with hexane=methylene chloride-isopropanol mixtures.

Amino Acids↗

PubMed1975

Open-flask alkaline hydrolysis of amino acid phenylthiohydantoin derivatives.

It is suggested that the open-flask system for the acid hydrolysis of proteins described earlier can also be used to advantage for the alkaline hydrolysis of amino acid phenylthiohydantoin derivatives obtained in protein sequential analysis according to Edman. Results obtained under nitrogen and argon atmospheres are reported for the hydrolysis of 0.5 mumole of each phenylthiohydantoin derivative, which is the amount of protein that is usually employed in an automatic sequenator.

Alkalies↗

PubMed1999

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.

Acetonitriles↗

PubMed1975

The amino-acid sequence of isoinhibitor K form snails (Helix pomatia). A sequence determination by automated Edman degradation and mass-spectral identification of the phenylthiohydantoins.

Performic-acid-oxidized isoinhibitor K of snails (Helix pomatia) was subjected to arginine-directed tryptic proteolysis. Six peptide fragments including one overlap peptide from limited cleavage of the Arg-3-Pro-4 bond were purified to homogeneity. Four arginine peptides and the C-terminal peptide were sequenced by automatic Edman degradation using a special peptide program. The phenylthiohydantoins were all identified by chemical ionization mass spectrometry, except four cysteic acid residues that were identified on an amino acid analyzer after acid hydrolysis. Quantitative evaluation of the phenylthiohydantoins by chemical ionization mass spectrometry using total molecular-ion beam integration greatly facilitated sequencing. The mass spectrum of the dipeptide less than Glu-Gly revealed that the N-terminus was blocked by pyroglutamic acid. The complete amino acid sequence of isoinhibitor K was determined. An almost 50% homology between the sequences of the snail inhibitor and the bovine trypsin-kallikrein inhibitor (Kunitz) became obvious. A comparison of all homologous sequences of this particular class of proteins known to date is presented.

Amino Acid Sequence↗

PubMed1990

Effects of methanol and urea on optical resolution of phenylthiohydantoin-DL-amino acids by micellar electrokinetic chromatography with sodium N-dodecanoyl-L-valinate.

Effects of methanol and urea on enantiomeric resolution of phenylthiohydantoin (PTH)-DL-amino acids by micellar electrokinetic chromatography with sodium N-dodecanoyl-L-valinate (SDVal) were investigated. PTH-DL-Met that could not be optically resolved with an SDVal solution alone was resolved by using an SDVal/methanol solution. Four PTH-DL-amino acids were separated from each other and each pair of enantiomers was also optically resolved, although seriously tailed peaks were observed for each solute. The addition of urea to SDVal/methanol solutions improved these peak shapes.

Amino Acids↗

PubMed2004

Separation of phenylthiohydantoin amino acids by capillary electrochromatography.

Capillary electrochromatography (CEC) was employed as a rapid and high-efficiency method for the isocratic separation of all 20 important phenylthiohydantoin (PTH) amino acids, the end products of Edman degradation during N-terminal protein sequencing. For this purpose, 75 microm ID fused-silica capillaries were packed with standard 3 microm Hypersil octadecyl silica (ODS) particles using a two-step column fabrication process, which represents a fast, reliable and efficient means of producing long-term stable columns. The influence of solvent composition, pH, type of buffer cation, buffer concentration, and temperature on retention behavior of PTH amino acids was investigated. Same-day and day-to-day reproducibility of the retention times (over a period of two months) were found to be better than 3%. When comparing this new technique with traditional reversed phase-high performance liquid chromatography (RP-HPLC) methods applied in automated protein sequenators, CEC shows essentially shorter separation times and superior resolution.

Acetonitriles↗

PubMed1995

A simple method for differentiating Leu and Ile in peptides. The negative-ion mass spectra of [M-H]- ions of phenylthiohydantoin Leu and Ile.

Phenylthiohydantoin (PTH) amino acid derivatives are formed during sequential Edman degradation of peptides and proteins. Isomeric Leu and Ile (which differ only in respectively having iso-butyl and sec-butyl chains, and are often difficult to distinguish by conventional mass spectrometric techniques) may be readily identified by the characteristic decompositions of the [M-H]- ions of their PTH derivatives. The Leu spectrum shows major loss of propane, while that of the Ile derivative shows elimination of both methane and ethane. This method may be used routinely with 10 microgram quantities of peptide material.

Isoleucine↗

PubMed2002

Rapid separation of phenylthiohydantoin amino acids: ambient pressure ion-mobility mass spectrometry (IMMS).

An electrospray ionization (ESI) ambient pressure ion-mobility spectrometer (APIMS) interfaced to an orthogonal reflector time-of-flight mass spectrometer (TOFMS) was evaluated for the first time as a detector for the identification of phenylthiohydantoin (PTH)-derivatized amino acids, the final products in the Edman sequencing process of peptides and proteins. The drift and flight times of the twenty common PTH amino acids were characterized by a well-defined 2-D mobility/mass spectral pattern. The combination of mobility/mass modes of analysis gave rise to a unique trend-line formation for the series of PTH amino acids. In addition, each PTH amino acid had a unique reduced mobility constant K(o), thus enabling the differentiation of all the amino acid derivatives including the PTH-leucine and PTH-isoleucine isomers. More importantly it was shown that it was possible to resolve a complete reference mixture of PTH amino acids in a single experimental run in less than 1 min. Detection limits for the PTH amino acids were found to range from 1.04 to 3.52 ng; indicating that the limits of detection were less than 17.0 pmol for all of the PTH amino acids.

Amino Acids↗

PubMed1978

The effect of thiodiglycol and dithiothreitol on the alkaline hydrolysis products of certain amino acid phenylthiohydantoins.

The thiazolinone and phenylthiohydantoin derivatives of most amino acids can be hydrolyzed with alkaline dithionite to generate the free amino acid. The acidification of this hydrolysate with 3 N HCl containing thiodiglycol leads in the case of glutamic acid, glutamine, aspartic acid, asparagine, and S-carboxy-methylcysteine to the generation of ninhydrin-reacting components having the chromatographic properties of other amino acids. The use of dithiothreitol instead of thiodiglycol appears to be more satisfactory in most instances.

Amino Acid Sequence↗

PubMed1985

Synthesis of side chain-protected amino acid phenylthiohydantoins and their use in quantitative solid-phase Edman degradation.

Solid-phase Edman degradation of synthetic peptidyl-resins has been used advantageously to detect errors of deletion which might occur during Merrifield peptide synthesis. To facilitate complete quantitation of the resulting phenylthiohydantoin(PTH)-amino acids, the PTH derivatives of the following side chain-protected amino acid residues have been synthesized: Arg(Tos), Asp(OBzl), Cys(3,4-(CH3)2-Bzl), Glu(OBzl), Lys(2-ClZ), Ser(Bzl), Thr(Bzl), Tyr(2-BrZ), and Tyr(2,6-Cl2Bzl). For each derivative, a melting point, elemental analysis, and extinction coefficient were obtained. With these new compounds as HPLC standards, an unequivocal assignment and quantification of each side chain protected amino acid was possible. A quantitative analysis was performed for six model peptides with the general formula Ala-X-Leu-Y-Ala-Gly-NHCH2-resin (where X and Y represented different side chain-protected amino acyl residues). We have found solid-phase Edman degradation to be a useful aid for the characterization of peptides when they are used unpurified as synthetic antigens.

Amino Acid Sequence↗

PubMed1986

Separation of amino acid phenylthiohydantoin derivatives by high-pressure liquid chromatography.

Separation of the phenylthiohydantoin (PTH) derivatives of all 20 common amino acids is accomplished in approximately 11 min with excellent resolution by using high-pressure liquid chromatography. The chromatography is achieved at 50 degrees C on an Altex reversed-phase PTH-C18 column in an ammonium acetate-buffered acetonitrile, pH 4.5, mobile phase. Simple isocratic and linear gradient steps are used. Retention times for the various PTH-amino acids are very reproducible. Because the baseline is flat and free of background noise, PTH-amino acids can be detected in the low picomole range. The simplicity of this chromatographic system allows it to be easily automated.

Amino Acid Sequence↗

PubMed1987

Rapid resolution of phenylthiohydantoin amino acids by thin-layer chromatography on silica gel plates impregnated with transition metal ions.

The resolution of a 15-component mixture of phenylthiohydantoin (PTH) amino acids using metal ion impregnated silica gel plates is reported. The spots are located by exposing the chromatograms to an iodine chamber. The method provides a rapid, simple, and less expensive chromatographic system, provides resolution for certain difficult combinations, and leaves the PTH amino acids unaltered chemically.

Amino Acids↗

PubMed1988

Analysis of phenylthiohydantoin amino acid mixtures for sequencing by thermospray liquid chromatography/mass spectrometry.

Phenylthiohydantoin (PTH) amino acids, the derivatives of amino acids liberated in the course of automated N-terminal sequence analysis of peptides and proteins, are most commonly identified by high-performance liquid chromatography. This communication describes an extension to the methodology for PTH amino acid identification which exploits thermospray liquid chromatography/mass spectrometry for use in the confirmation of PTH amino acid identifications previously made solely on the basis of retention times. Thermospray mass spectra of the 19 synthetic PTH amino acids corresponding to the residues commonly observed during N-terminal sequencing have been acquired. These spectra show strong signals for the protonated molecular ion, accompanied in several cases by ions produced by limited fragmentation of the amino acid side chain and/or the PTH ring system. A reverse-phase separation protocol has been adapted for use with thermospray. The method permits recognition of the protonated molecular ions of all the standard PTH amino acids at the 150-pmol level on the basis of signal-to-noise ratios of 10:1 or better with full scanning. The method has been tested on the N-terminal amino acid sequence analysis of 200 pmol of the standard protein beta-lactoglobulin A, and has been found useful in the study of selected side-products of the sequencing chemistry.

Amino Acid Sequence↗

PubMed1991

Biotinylated peptides/proteins. II. Identification of biotinylated lysyl phenylthiohydantoins.

The identification and characterization of biotinylated lysyl residues in a polypeptide chain by automated sequence analysis is described. An in depth analytical study was conducted for the delineation of N epsilon modification of lysyl residues with N-hydroxysuccinimide esters of biotin and 6-aminohexanoic biotin. Confirmation of the structure of the phenylthiohydantoin derivatives of N epsilon biotinylated lysine was achieved by mass spectrometry. The analytical study focused on the identification of biotinylated lysine-4 of neuropeptide Y which served as a model peptide for the analytical procedures detailed.

Amino Acid Sequence↗

PubMed1992

The development of high-performance liquid chromatographic analysis of allyl and allyloxycarbonyl side-chain-protected phenylthiohydantoin amino acids.

Ten phenylthiohydantoin (PTH) amino acids possessing allyl (Al) or allyloxycarbonyl (Aloc) side-chain-protecting groups have been characterized by high-performance liquid chromatography for use in Edman degradation sequence analysis. Optimized separation of side-chain-protected and -unprotected PTH amino acids was achieved on a C-18 reversed-phase column with a two-step gradient spanning 32 min. Five of the side-chain-protected amino acids [Cys(Al), Cys(Aloc), Lys(Aloc), Thr(Aloc), Tyr(Al)] were completely stable to the conditions of PTH derivatization, four [Asp(OAl), Arg(Aloc)2, Glu(OAl), Ser(Aloc)] were partially deprotected during PTH derivatization, and one [His(Aloc)] was completely deprotected during PTH derivatization. All allyl-based derivatives were well resolved from their side-chain-unprotected counterparts. Studies on the stability to piperidine treatment showed Asp(OAl), Cys(Al), Glu(OAl), Lys(Aloc), Thr(Aloc), and Tyr(Al), and possibly Arg(Aloc)2 and Ser(Aloc), to be suitable for peptide synthesis by 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry. Edman degradation of Al and Aloc side-chain-protected Conus geographus Lys9-alpha-conotoxin GI synthesized on 4-methylbenzhydrylamine-copoly(styrene-1%-DVB)-resin demonstrated the usefulness of these derivatives for solid-phase preview sequence analysis.

Amino Acid Sequence↗