Detection of peptidyl-3,4-dihydroxyphenylalanine by amino acid analysis and microsequencing techniques

3,4-Dihydroxyphenylalanine (DOPA) is an amino acid that occurs naturally in the primary sequence of many proteins and peptides. Detection of peptidyl-DOPA, however, can be elusive. This is due (i) to its coelution with leucine on most of the ion exchangers used in amino acid analysis and (ii) to the coelution of phenylthiohydantoin (PTH)-DOPA with PTH-alanine during routine C18 reversed-phase HPLC following automated Edman degradation. By application of appropriately timed temperature and/or gradient modifications during chromatography, DOPA and its PTH derivative can be adequately resolved for detection by both amino acid analysis and gas-phase sequencing.     

Isocratic separation of PTH-amino acids at picomole level by reverse-phase HPLC in the presence of sodium dodecylsulfate

The phenylthiohydantoin (PTH) derivatives of protein amino acids have been separated by reverse-phase high performance liquid chromatography (HPLC) on a fully end-capped C18 column using an isocratic solvent system. The developing solvent was 0.01 M sodium acetate buffer (pH 4.5) containing 39.5% acetonitrile and 0.02% sodium dodecylsulfate (SDS). With an automated liquid chromatography equipped with a dual-channel detector, operating at 254 and 313 nm, the present isocratic separation system was quite useful for routine microanalysis of PTH-amino acids released with a "gas-phase" sequencer. The time for one run was approximately 23 min and the limit of analysis approximately 2.5 pmol of a PTH-amino acid.     

High-performance liquid chromatography of side-chain-protected amino acid phenylthiohydantoins

Eighteen side-chain-protected amino acids, routinely employed in solid-phase peptide synthesis, were derivatized to their phenylthiohydantoins (PTH) by one cycle of the Edman degradation. All of these side-chain-protected PTH amino acids elute, with almost-baseline resolution, in less than 18 min by high-performance liquid chromatography, utilizing a biphasic gradient of acetonitrile in 0.01 n sodium acetate, pH 4.5, or a linear gradient of 0 to 100% acetonitrile with the exception of the coelution of a O-benzyl-threonine and carbobenzoxy-lysine phenylthiohydantoin amino acids. The derivatized amino acids were subjected to reverse-phase chromatography on a Zorbax ODS column and monitored at 254 nm. None of the PTH amino acids coelute with side-chain-protected PTH amino acid counterparts, although PTH-tosyl-histidine undergoes deprotection to PTH-histidine in the Edman degradation. A protected decapeptide attached to a chloromethylated polystyrene resin was degraded on a solid-phase sequencer in 16 h. The PTH amino acids resulting from the automated Edman degradation on the decapeptide were fully resolved and quantified in less than 3 h demonstrating that automated high-performance liquid chromatography can keep pace with both the automated sequencer and synthesizer which requires minimally 2–3 h for attachment of each residue to the growing peptide chain.     

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.     

Detection of methylated asparagine and glutamine residues in polypeptides

A residue of gamma-N-methylasparagine (gamma-NMA) is found at position beta-72 of many phycobiliproteins. delta-N-Methylglutamine is present in some bacterial ribosomal proteins. gamma-NMA was synthesized by reacting the omega-methyl ester of aspartate with methylamine and delta-N-methylglutamine by reaction of pyroglutamate with methylamine. These derivatives and the omega-methyl esters of aspartate and glutamate were characterized by melting point, by thin-layer chromatography, by amino acid analysis, by NMR spectroscopy, and after conversion to the phenylthiohydantoin (PTH) derivative. The gamma-NMA residues in peptides from allophycocyanin, C-phycocyanin, and B-phycoerythrin were stable under the conditions of automated sequential gas-liquid phase Edman degradation. On HPLC, PTH-gamma-NMA co-eluted with PTH-serine and was accompanied by a minor component eluting just prior to dimethylphenylthiourea. Similar results were obtained on manual derivatization of synthetic gamma-NMA to prepare the PTH derivative. The PTH-delta-N-methylglutamine standard eluted near the position of dimethylphenylthiourea under the usual conditions employed for the identification of PTH-amino acid derivatives in automated protein sequencing.     

Covalent attachment of peptides for high sensitivity solid-phase sequence analysis

We have developed a method for the high efficiency covalent immobilization of picomole to nanomole quantities of peptides in a form compatible with high sensitivity gas-liquid or solid-phase sequence analysis. Glass fiber filter paper was derivatized with amino-phenyltriethoxysilane and peptides were applied to circular disks cut to 1-cm diameters. Peptides were covalently immobilized on the aminophenyl-glass fiber paper through their terminal alpha-carboxyl groups and amino acid side-chain carboxyl groups by activation with the water-soluble reagent N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Disks containing the covalently attached peptide were directly inserted into the cartridge of an automated sequenator for sequence analysis by the Edman degradation. Peptides prepared in this way could be routinely sequenced through to and including the C-terminal amino acid residue, at extraordinarily low backgrounds. The covalent immobilization of peptide fragments allowed far more flexibility in sequencing conditions, including the use of polar extraction solvents to increase the yield of phenylthiohydantoin (PTH)-His and PTH-Arg and the use of alternative Edman-type sequencing reagents with enhanced detectability, such as the chromophoric compound 4- (N,N'-dimethylamino)azobenzene-4'-isothiocyanate. The potential of this high efficiency immobilization method for contributing to the development of sequencing chemistries with enhanced sensitivity is discussed.     

Accelerated high-sensitivity microsequencing of proteins and peptides using a miniature reaction cartridge.

Greatly reduced automated protein sequencing degradation times of less than 25 min with concurrent on-line phenylthiohydantoin (PTH) derivative analysis times of less than 16 min have been achieved by using a miniaturized reaction cartridge with optimized chemical and analytical cycles. Using this method a wide range of standard and novel peptides and proteins have been sequenced with reproducibly high initial and repetitive cycle yields. In these accelerated analyses the recovery of the more labile PTH derivatives was markedly improved by using elevated pressure during cleavage steps and temperature programming throughout the Edman cycle.     

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.     

Identification of unusual amino acids in peptides using automated sequential Edman degradation coupled to direct detection by electrospray-ionization mass spectrometry

The determination of the primary structure of peptides and proteins is routine in many laboratories; however, many of the obtained sequences are incomplete or can be misinterpreted when the samples contain unusual amino acids. Here we report the development of an automated peptide sequenator coupled to an electrospray-ionization (ESI) mass spectrometer (MS) that, in conjunction with minor modifications to the sequencing conditions and, in some cases, prior derivatization of amino acids, allows the detection of the phenylthiohydantoin (PTH) derivatives of a number of unusual amino acids. Using the coupled sequenator-ESI-MS system we were able to determine the complete sequence of the lantibiotic gallidermin, a partial sequence of the calcium-dependent peptide antibiotic CDA2 as well as the pool sequence of a mixture of synthetic peptides containing nonproteinogenic amino acids. In addition to the 20 proteinogenic amino acids, the procedure was able to detect PTH derivatives of hydroxyphenylglycine, 2,3-didehydroasparagine, 3-methylglutamic acid, oxytryptophan, ornithine, N-methylglycine, dihydroxyphenylalanine, and alpha-aminoisobutyric acid. Similarly, after a simple derivatization procedure, we were also able to correctly identify educts of 2,3-didehydroalanine, 2,3-didehydrobutyrine, lanthionine, and 3-methyllanthionine.     

Separation of o-phthalaldehyde derivatives of amino acids of the internal pool of yeast by reverse-phase liquid chromatography

Summary Derivatization of primary amino acids with orthophthalaldehyde and -mercaptoethanol forms derivatives that can be detected by absorbance at 340 nm. These were separated by reverse-phase high performance liquid chromatography (HPLC). A step- or more complex gradient (acetonitrile/phosphate buffer gradient) was used. The effects of several parameters (pH, ionic strength, etc) were characterized and used to design a rapid separation of the amino acids commonly found in physiological fluids. The method described is rapid, sensitive and precise as sensitivity limits are about 25 pmol and the separation time, injection to injection, is 16 min.     

Selective separation of tryptophan-containing peptides via hydrophobic modification with 2-nitro-4-carboxyphenylsulfenyl chloride

Selective separation of tryptophan-containing peptides has been attempted using 2-nitro-4-carboxyphenylsulfenyl chloride (NCps)-C1 as a reagent for hydrophobic modification of tryptophan. S-Carboxymethylated proteins were modified with NCps-C1 in 70% acetic acid and digested with TPCK-trypsin, and the digests were fractionated, directly or after partial fractionation on a Sephadex G-25 column, by high performance liquid chromatography using a reverse phase column. The tryptophan-containing peptides from the tryptic digests of S-carboxymethylated hen-egg white lysozyme and Trimeresurus flavoviridis phospholipase A2 were thus selectively separated and the amino acid sequences were determined, showing the validity of the method. The phenylthiohydantoin derivative of 2-(2'-nitro-4'-carboxyphenylthio)-L-tryptophan was synthesized and its spectroscopic and chromatographic properties determined, enabling us to identify the derivative on Edman sequencing.     

Direct identification of gamma-carboxyglutamic acid in the sequencing of vitamin K-dependent proteins.

We report the first direct method for the identification of the vitamin K-dependent Ca2+ binding amino acid, gamma-carboxyglutamic acid (Gla), in the sequencing of proteins. The carboxyl groups on the protein are first converted to methyl esters with methanolic HCl, a procedure that reduces the polarity of the resulting ATZ derivative of dimethyl-Gla and so greatly improves its extraction from the polybrene-treated glass fiber filter. After conversion to the PTH derivative in methanolic HCl, the resulting dimethyl ester of PTH Gla can be identified directly by a simple modification of the standard HPLC program for the separation of PTH derivatives. This methylation procedure can be used to identify Gla residues in proteins bound to PVDF membranes, as we demonstrate for matrix Gla protein and prothrombin, and to evaluate directly the degree of partial gamma-carboxylation at given glutamic acid residues, as we demonstrate for the 50% gamma-carboxylation of residue 17 in human bone Gla protein.     

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.     

Examination of automated polypeptide sequencing using standard phenyl isothiocyanate reagent and subpicomole high-performance liquid chromatographic analysis

The feasibility of accurate protein sequencing at the subpicomole level, using automated Edman chemistry and "on-line" HPLC analysis, was studied. Several modifications of the standard system were first introduced. A larger portion of the phenylthiohydantoin amino acids (70%) is analyzed. Dissolution in 10% acetonitrile is improved by short periodic bursts of argon. Losses on the column of subpicomole amounts of analytes, in the presence and absence of scavengers, were quantitated; they are related to destruction rather than to unspecific sticking to the stationary phase. Baseline drift, for a large part caused by the presence of ultraviolet absorbing N,N-dimethylphenylthiourea in solvent B, is completely eliminated by the addition of a twofold molar excess of tryptophan to solvent A. This allows real time recording of the 269-nm absorption detector signal at 0.0005 absorption unit full scale. The combined modifications result in an eightfold increase in sensitivity over standard methods. Sequence calling at the 2 to 10 pmol level, through visual inspection of chromatograms, becomes increasingly simple this way. Once the sequenceable signal drops below the 1 pmol level in the course of a run, meticulous comparison and matching of the preliminary calls with a spreadsheet of peak integration data are necessary for accurate assignments. Reliable sequencing, with signals at the subpicomole level, is now feasible for stretches of over 10 residues. Contaminating amino acids and polypeptides and incompletely removed reaction by-products constitute a major problem for analysis at this level. Future limits to sensitivity of Edman sequencing will primarily depend on improved micropreparations of proteins in cleaner environments, higher purity reagents and solvents, instrument miniaturization, and solid-phase techniques.     

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.     

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.     

High-sensitivity phenylthiohydantoin amino acid analysis using conventional and microbore chromatography

Reversed-phase microbore high-performance liquid chromatography was investigated for high-sensitivity analysis of phenylthiohydantoin (PTH) amino acids. A mixed nitrile alkylsilane bonded phase was developed and ternary gradient elution conditions were devised for resolution 150 × 4.6 mm I.D. column and transferred to a 150 × 1 mmI.D. microbore column. The performance of these columns was evaluated in terms of PTH amino acid resolution, enhanced sample detectability, and retention time precision. For this work a general purpose high-performance liquid chromatograph was modified to reduce extra column band broadening and a preformed gradient elution technique was developed to achieve rapid analysis times at microbore flow-rates. The microbore high-performance liquid chromatographic system is useful for high-sensitivity analysis of PTH amino acids in micro-sequencing applications.     

EDMAN SEQUENCING RESEARCH GROUP 2004 STUDY: MODIFIED AMINO ACIDS IN EDMAN SEQUENCING

Edman degradation sequencing relies on comparing high-performance liquid chromatography retention times of the sample phenylthiohydantoin amino acids with phenylthiohydantoin amino acid standards. The elution characteristics of the twenty common amino acids have been well characterized, which aids in making confident assignments. Modified amino acids may present more of a challenge since they are not part of the commonly used standards and because the protein sequencer analyst may not have experience with them. Laboratories requesting a sample were sent a tube containing approximately 775 pmoles of a 20-amino-acid synthetic peptide composed of several modified amino acids that may be found in proteins or are generated during sample preparation. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the Edman Sequencing Research Group website (ESRG). The goal of the study consisted of two parts: assessment of the ability to correctly assign all the amino acids in the peptide, including the modified amino acids; and the collection and compiling of elution time characteristics of modified amino acids for instruments used in the study. The resulting compilation of the modified amino acid elution times and running conditions will be accessible at the Association of Biomolecular Resource Facilities (ABRF) ESRG website for future reference. The ABRF ESRG 2004 sample is the 16th in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.     

Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support

A method allowing initial sequencing yields of 60-85% to be consistently obtained from samples prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer is described in detail. Conducting electrophoresis at a pH near neutrality is the single most important of the modifications made to earlier procedures, but pre-electrophoresis in the presence of glutathione or sodium thioglycolate and use of Immobilon polyvinylidene difluoride membranes all contribute to the success of the technique. When tryptophan was the NH2 terminus of a protein, the phenylthiohydantoin (PTH)-derivative recovered appeared to be an irreversible oxidation product if pre-electrophoresis was not performed. Following pre-electrophoresis, the PTH-derivative recovered co-migrated with that of unmodified tryptophan, and the recovery was higher. Recovery of methionine as its PTH-derivative was not affected by pre-electrophoresis suggesting that thioglycolate in the electrophoresis buffer during sample separation prevented or reversed oxidation of methionine sulfur but did not protect tryptophan.     

Microsequence analysis of peptides and proteins. IX. Manual gas-phase microsequencing of multiple samples

A novel apparatus for performing manual gas-phase Edman chemistry on protein and peptide samples is described. Edman chemistry is performed in 6 to 10 Teflon continuous flow reactors (CFR), previously described by J.E. Shively et al. (1987) Anal. Biochem. 163, 517-529). The CFRs are packed with 10-15 mg of Polybrene-coated spherical silica (Porasil B, Waters Associates). The gas-phase coupling reagent and cleavage reagent are 5% aqueous triethylamine and anhydrous trifluoroacetic acid, respectively, delivered by a stream of argon gas. The delivery of the gas-phase reagents is manually controlled with Hamilton 3-way valves and 2-way valves, and that of the solvents, ethyl acetate and butyl chloride, by syringe pipetting. The average cycle time is 15-20 min for 6 to 10 samples run simultaneously. Conversion of the anilinothiazolinone to phenylthiohydantoin (PTH) amino acid derivatives is accomplished manually with 25% aqueous trifluoroacetic acid. The PTH amino acids are analyzed by reversed-phase HPLC using an autosampler for handling multiple samples. Excellent results were obtained in the 100-200 pmol range. Protein samples can be sequenced from 15-20 cycles, and peptide samples usually to the COOH terminus. Initial yields ranged from 30 to 60% and repetitive yields ranged from 90 to 96%. The sample washout and size of background peaks are significantly reduced, compared to older methods of manual sequence analysis. The yields and background signal to noise are comparable to automated gas-phase Edman chemistry. The improved manual Edman described represents a low cost alternative to automated sequence analysis, and has the advantage being able to process multiple samples simultaneously.