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.     

Sensitization of Edman amino acid derivatives using the fluorescent reagent, 4-aminofluorescein

The ability to analyze amino acid derivatives at the femtomole level is one of the most interesting challenges in the field of protein microsequencing. 2-Anilino-5-thiazolinone amino acids, obtained by Edman degradation, were quantitatively derivatized with fluorescent primary amines. The most fluorescent reagent tested was 4-aminofluorescein. The amino acid derivatives sensitized with this reagent were separated using reversed-phase high-performance liquid chromatography and identified at the 100 attomole level. Incorporation of this method into the operation of a conventional automated sequencer is also described.     

Isolation and amino acid sequence of the TRH-potentiating peptide from bovine hypothalamus.

A neuropeptide termed TRH-potentiating peptide, which potentiates TRH-evoked thyrotropin secretion by antehypophysis in vitro, was isolated from an acetonic powder of bovine hypothalamus. The peptide was purified to homogeneity by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography and reverse phase high performance liquid chromatography. The complete amino acid sequence of the decapeptide was determined as Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr by automated Edman degradation with a solid-phase sequencer. Bovine TRH-potentiating peptide is structurally identical to Ps4, a decapeptide which was deduced from the cDNA encoding the rat TRH precursor. This study provides for the first time a direct chemical evidence for the existence of non-TRH peptides originating from posttranslational processing of the TRH precursor in vivo.     

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.     

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.     

Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin

A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.     

Solid-phase sequencing on the gas-phase sequencer

Automated Edman degradation has been successfully used for determining the primary structure of numerous peptides and proteins. Quantitative solid-phase Edman degradation has great potential use for amino acid sequence analysis of synthetic peptides assembled on resin support by the Merrifield procedure. We report here the combined use of a modified gas-phase sequencer program and our improved reversed-phase HPLC analysis for PTH-amino acids to carry out the sequence analysis on synthesized peptide resins. This approach is far more sensitive than using glass beads on the conventional solid-phase sequencer. The peptide was assembled on copoly (styrene-1% divinylbenzene) resin beads at an initial substitution of 0.54 mmol/g. On a routine basis, 10-15 resin beads are used, and a repetitive yield of 94% is obtained: as few as 4 beads can be successfully sequenced. The HPLC PTH-amino acid analysis is sensitive down to subpicomole quantities. This procedure offers a sensitive and rapid analytical tool for checking the purity of peptides as they are being assembled on solid support.     

Automatic Edman microsequencing of peptides containing multiple unnatural amino acids

It is now routine using automatic Edman microsequencing to determine the primary structure of peptides or proteins containing natural amino acids; however, a deficiency in the ability to readily sequence peptides containing unnatural amino acids remains. With the advent of synthetic peptide chemistry, combinatorial chemistry, and the large number of commercially available unnatural amino acids, there is a need for efficient and accurate structure determination of short peptides containing many unnatural amino acids. In this study, 35 commercially available alpha-unnatural amino acids were selected to determine their elution profile on an ABI protein sequencer. Using a slightly modified gradient program, 19 of these 35 PTH amino acids can be readily resolved and distinguished from common PTH amino acids at low picomole levels. These unnatural amino acids in conjunction with the 20 natural amino acids can be used as building blocks to construct peptide libraries, and peptide beads isolated from these libraries can be readily microsequenced. To demonstrate this, we synthesized a simple tripeptide "one-bead one-compound" combinatorial library containing 14 unnatural and 19 natural amino acids and screened this library for streptavidin-binding ligands. Microsequencing of the isolated peptide-beads revealed the novel motif Bpa-Phe(4-X)-Aib, wherein X = H, OH, and CH3.     

Fluorescence detection of amino acids in the postcleavage conversions for manual sequencing of a peptide

A modified Edman degradation method where fluorescent derivatives of amino acids were generated from the postcleavage products of a peptide is described. In the method, the target peptide was applied onto double glass fiber membranes in a small filter disk (4 mm i.d.) and then treated with small amounts of reagents for the manual sequencing of the peptide. The anilinothiazolinone (ATZ) of N-terminus amino acid residue after the isolation from the solid-phase membranes was reacted with a primary amine, 4-(1′-cyanoisoindolyl)aniline (CIA), to form a more stable and sensitive fluorescent derivative, phenylthiocarbamoyl-CIA. An average yield of 85% was obtained in neutral pH conditions for the CIA reaction. The ATZ-CIA-amino acids were separated by reversed-phase liquid chromatography and detected by fluorometry. The lower limits of the detection for amino acids after the Edman degradation were 0.16 to 0.52 pmol (signal/noise ratio = 3) on the column. The sensitivity was approximately 10 times higher than ultraviolet absorbance detection of phenylthiohydantoin products in the conventional Edman degradation. The suitability of the method was demonstrated by the sensitive manual sequencing of insulin chain B composed of 30 amino acids.     

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.     

Isolation and sequence of tryptic peptides from the proton-pumping ATPase of the oat plasma membrane

In crude extracts of plant tissue, the M_r = 100,000 proton-pumping ATPase constitutes less than 0.01% of the total cell protein. A large-scale purification procedure is described that has been used to obtain extensive protein sequence information from this enzyme. Plasma membrane vesicles enriched in ATPase activity were obtained from extracts of oat roots by routine differential and density gradient centrifugation. Following a detergent wash, the ATPase was resolved from other integral membrane proteins by size fractionation at 4°C in the presence of lithium dodecyl sulfate. After carboxymethylation of cysteine residues and removal of detergent, the ATPase was digested with trypsin and resultant peptide fragments separated by reverse phase high performance liquid chromatography. Peptides were recovered with high yield and were readily sequenced by automated Edman degradation on a gas-phase sequencer. Of the eight peptides sequenced, six showed strong homology with known amino acid sequences of the fungal proton-pumping and other cation-transporting ATPases.     

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.     

Primary structure of PDC-109, a major protein constituent of bovine seminal plasma

The two major protein components of bovine seminal plasma, PDC-109 and BSP I, have been purified by gel filtration, partition chromatography and reverse-phase high performance liquid chromatography from an 86% ethanol precipitate of bovine seminal plasma ejaculate. The complete 109-residue amino acid sequence of PDC-109 has been established by automated Edman degradation of the intact peptide as well as its proteolytic digestion and cyanogen bromide cleavage fragments. The 12,774 dalton structure has two structurally similar domains of 38 and 41 amino acids, each containing two disulfide bonds.     

Amino acid sequence of human plasma apolipoprotein C-III from normolipidemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-III (apoC-III) isolated from normal subjects is described. ApoC-III is a linear polypeptide chain of 79 amino acids. Tryptic digestion of intact apoC-III produced 5 major peptides, while tryptic digestion of the citraconylated protein yielded two peptides. The complete amino acid sequence of apoC-III was determined by the automated Edman degradation of the intact protein as well as the various tryptic peptides. Phenylthiohydantoin amino acids were identified by high-performance liquid chromatography and chemical ionization mass spectrometry. The amino acid sequence of apoC-III isolated from normolipidemic subjects is identical to the apoC-III sequence derived from the cDNA sequence and differs at 4 positions from the previously reported sequence of apoC-III derived from a patient with type V hyperlipoproteinemia.     

Purification and cloning of a novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin-B was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY by Edman degradation. The cDNA encoding ixosin-B was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 89 amino acids including mature ixosin-B. Purified ixosin-B exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide. It is also the fourth family of antimicrobial peptide from hard ticks.     

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.     

Amino acid sequence of the phosphorylation site of isocitrate dehydrogenase fromEscherichia coli

InEscherichia coli, NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) may undergo a phosphorylation catalyzed by a cAMP-independent protein kinase, with a concomitant decrease in catalytic activity. In this report, we describe the purification and amino acid sequence of a³²P-labeled peptide obtained from in vivo³²P-labeled isocitrate dehydrogenase. The³²P-labeled peptide was isolated from a tryptic digest and found to contain seven amino acids, including a single serine residue. Following automated Edman degradation and reversephase high-pressure liquid chromatography of the phenylthiohydantoin-amino acids, the sequence of this peptide was established to be-Ser(P)-Leu-Asn-Val-Ala-Leu-Arg.     

Separation of dimethylaminoazobenzenethiohydantoin amino acids by high-performance liquid chromatography at low picomole concentrations

The separation of all common dimethylaminoazobenzenethiohydantoin (DABTH) amino acids derived from modified Edman sequencing can be achieved by using high-performance liquid chromatography. All derivatives, including DABTH-Ile and DABTH-Leu, can be readily separated in a solvent mixture of sodium acetate buffer and 1% ethylene dichloride in acetonitrile. The high absorbance of the DABTH amino acids at 436 nm makes possible the quantitative determination of these derivatives at picomole concentrations in a relatively short time (30–40 min).     

A novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as GLHKVMREVLGYERNSYKKFFLR by Edman degradation and its molecular weight was 2870.5 analyzed by fast atom bombardment (FAB) mass spectrometry. This is the first antimicrobial peptide from ticks that lacks cysteine in its primary structure. The cDNA encoding ixosin was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 79 amino acids including mature ixosin. Purified ixosin exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide.     

The primary structure of the Chloroflexus aurantiacus reaction-center polypeptides

The complete nucleotide sequence of two Chloroflexus aurantiacus reaction-center genes has been obtained. The amino acid sequence deduced from the first gene showed 40% similarity to the L subunit of the Rhodobacter sphaeroides reaction center. This L subunit was 310 amino acids long and had an approximate molecular mass of 35 kDa. The second gene began 17 bases downstream from the first gene. The amino acid sequence deduced from it (307 amino acids; 34950 Da) was 42% similar to the M subunit of the Rhodobacter sphaeroides reaction center. 20% of the deduced primary structure were confirmed through automated Edman degradation of cyanogen bromide peptide fragments or N-chlorosuccinimide peptide fragments isolated from the purified reaction-center complex or from the individual subunits. The peptides were isolated by preparative gel electrophoresis combined with molecular sieve chromatography in the presence of a mixture of formic acid, acetonitrile, 2-propanol and water. This method appeared to be applicable to the isolation of other hydrophobic proteins and their peptides.