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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↗

PubMed2001

Chemical C-terminal protein sequence analysis: improved sensitivity, length of degradation, proline passage, and combination with edman degradation.

Use of a C-terminal sequencer with modified solvents, reagent concentrations, chromatographic parameters, temperatures, and reaction cartridge geometry yields four sets of improvements in chemical degradations. They are increased sensitivity, longer runs, passage of Pro residues, and practical combination with N-terminal degradation. Over 200 proteins and protein fragments with sizes between 20 and 600 residues were analyzed. C-terminal sequences could be interpreted for more than 10 residues at high picomole sample levels, while the 10-pmol level gave 4-5 residues. The average initial yield was 15% but up to 30% could be achieved. The improved performance allowed combination of C- and N-terminal degradations from the same sample application. After initial Edman degradation, the sample is moved to the C-terminal instrument for continued sequencing. Proteins available in limited amount are thereby efficiently analyzed. Lys, modified from the N-terminal degradation, may be detected as the alkylated thiohydantoin-phenylthiocarbamyl-Lys derivative in the C-terminal degradation. Notably, C-terminal sequence analysis could be proceeded through Pro residues which unexpectedly were no absolute hindrance. The improved technique provides characterization of truncation patterns and microheterogeneities in proteins down to the 10-pmol level and is a useful approach for analysis of N-terminally blocked polypeptides.

Mass Spectrometry↗

PubMed1981

Quantitative solid-phase Edman degradation for evaluation of extended solid-phase peptide synthesis.

Quantitative solid-phase Edman degradation was used for the amino acid sequence analysis of synthetic peptidyl-resins prepared by the Merrifield solid-phase procedure. A model peptide, Ala-[3H]Pro-Ala-Gly-Phe-Ala-Gly-, was synthesized on a solid support and was sequenced to measure the efficiency of the solid-phase sequencing protocol used. An average of 92% of the first four residues was removed from the peptidyl-resin as indicated by subtractive amino acid analysis. Quantitation of the radioactive proline residue at cycle 2 revealed that it was efficiently recovered both from the acid conversion procedure (99%) and also following high-pressure liquid chromatography of the phenylthiohydantoin (Pth) amino acid (88%). In order to facilitate identification and quantification of the side chain protected Pth amino acids, we prepared these derivatives and characterized them by high-pressure liquid chromatography. Thereafter, by the use of solid-phase Edman degradation as an analytical procedure, the synthesis of residues 2-118 of the heavy-chain variable region (VH) of a homogeneous rabbit antibody was undertaken. At 10-15-residue intervals during the solid-phase synthesis, samples of peptidyl-resin were removed from the synthesis vessel and sequenced. When gross synthetic errors caused by deletion of amino acids residues were detected, the solid-phase synthesis was terminated and restarted by using modified protocols. A 117-residue peptidyl-resin was prepared finally which possessed the desired amino acid sequence as indicated by a series of solid-phase Edman degradation experiments. In the final degradation experiment on the 117-residue peptidyl-resin, a 92% efficiency for the automatic Edman reaction was measured ([3H]Leu, penultimate amino-terminal residue). We have found two advantages for the concurrent use of solid-phase Edman degradation during an extended solid-phase synthesis: (1) on the basis of the level of error due to incomplete incorporation of amino acids, the solid-phase assembly could be terminated in favor of restarting the synthesis, hence avoiding further work on a defective product and (2) direct verification of incorporation of amino acids, which during acid hydrolysis are destroyed (Cys, Trp) or are deamidated (Asn, Gln), is possible by high-pressure liquid chromatography of the corresponding Pth derivatives.

Amino Acid Sequence↗

PubMed2001

Assignment of the disulfide bonds of huwentoxin-II by Edman degradation sequencing and stepwise thiol modification.

A novel strategy combining Edman degradation and thiol modification was developed to assign the three disulfides of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of the spider Selenocosmia huwena. Phenylthiohydantoin (Pth) derivatives of Cys and the elimination product, dehydroalanine (DeltaSer), can be observed in the Cys cycles during Edman degradation of native HWTX-II. The appearance of two products indicates that the disulfides of HWTX-II were split and that the free thiol group of the second half cystine has been generated. Information about the nature of the disulfide bridges of HWTX-II could be obtained from the sequencing signal if the nascent thiols were modified stepwise by 4-vinylpyridine. Using this method the disulfide bridges of HWTX-II were assigned as Cys4-Cys18, Cys8-Cys29 and Cys23-Cys34, which is different from that seen in HWTX-I, a neurotoxic peptide from the same spider. Using this strategy, one can assign the disulfide bonds of small proteins by sequencing and modification n - 1 times, where n is the number of disulfide bonds in the protein. The above assignment of the disulfide bonds of HWTX-II was confirmed by MALDI-TOF MS of tryptic fragments of HWTX-II. Some disulfide interchanging during proteolysis was observed by monitoring the kinetics of proteolysis of HWTX-II by MALDI-TOF MS.

Amino Acid Sequence↗

PubMed2001

Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry.

Edman degradation remains the primary method for determining the sequence of proteins. In this study, accelerator mass spectrometry was used to determine the N-terminal sequence of glutathione S-transferase at the attomole level with zeptomole precision using a tracer of (14)C. The transgenic transferase was labeled by growing transformed Escherichia coli on [(14)C]glucose and purified by microaffinity chromatography. An internal standard of peptides on a solid phase synthesized to release approximately equal amounts of all known amino acids with each cycle were found to increase yield of gas phase sequencing reactions and subsequent semimicrobore HPLC as did a lactoglobulin carrier. This method is applicable to the sequencing of proteins from cell culture and illustrates a path to more general methods for determining N-terminal sequences with high sensitivity.

Carbon Radioisotopes↗

PubMed1990

N-terminal sequence determination of polypeptides and peptide mixtures by Edman degradation combined with californium-252 plasma desorption mass spectrometry.

The combination of manual Edman degradation with 252Cf plasma desorption mass spectrometry has been developed as an efficient method of polypeptide sequence determination. Results obtained with a variety of peptides and small proteins demonstrate unequivocal, two-fold sequence data, at each step from molecular weight identification of successively truncated Edman coupling and cleavage products. A rapid, greatly simplified procedure of manual Edman degradation with phenyl isothiocyanate was employed without extraction and purification steps, which was found compatible with plasma desorption mass spectrometry of low picomole amounts of peptide sample, at each cycle. Particular advantages of this combined method are the possibility of obtaining simultaneous sequence information from multicomponent peptide mixtures, such as proteolytic digests, and the direct identification of modified sequences, such as glycosylation sites. With the currently used conditions, plasma desorption mass spectrometry has been found feasible for approximately 30 sequence steps and sensitive to subnanomole amounts of proteins, with Mr up to approximately 10,000 Da.

Amino Acid Sequence↗

PubMed1976

Investigation of the covalent bond structure of peptide-matrix systems by Edman degradation of support-fixed peptides.

Two model peptides Ala-Ser-Asp-Tyr-Leu and Ala-Ser-Glu-Tyr-Leu have been synthesized and coupled with an without protecting groups to the amino groups of polystyrene resins. The yield in the binding operation was 93-97% by using water-soluble carbodiimides as condensing reagents. Edman degradation of the products and quantitative estimation of the amino acid phenylthio-hydantoins, as well as amino acid analysis of the partially and completely degraded resins, showed about 7-10% of the fixed peptides not to be involved in the degradation procedure. That part of the material which was not involved in the Edman degradation during the first step also remained inactive in the following degradation operations. The analytical data presented in the paper explain not only the course of the Edman degradation of peptides linked via the side-chain carboxylic groups to the resin, but they also describe the chemical reactivity of the various carboxylic groups in the binding operation. 22% of the peptides are bound to the resin by side-chain carboxylic groups in the case of the aspartate-containing peptide, 26-27% in the case of the glutamate-containing peptide. 75-77% and 72-75% respectively of the peptides are fixed to the resin via the C-terminal carboxylic group. No more than 3% of the fixed material may be bound bifunctionally to the support.

Amino Acids↗

PubMedDate not supplied

Edman degradation sequence analysis of resin-bound peptides synthesized by 9-fluorenylmethoxycarbonyl chemistry.

The efficacy of Edman degradation sequence analysis for evaluating the synthetic efficiency of peptide-resin assembly by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase methodology has been studied. Prior researchers have described the use of solid-phase "preview" sequence analysis for peptides synthesized by tertiary-butyloxycarbonyl (Boc) chemistry, where benzyl-based side-chain protecting groups and peptide-resin linkers are stable to the conditions of Edman chemistry. We have successfully sequenced a variety of resin-bound peptides synthesized by Fmoc chemistry, where tertiary-butyl-based side-chain protecting groups and peptide-resin linkers are labile to the conditions of Edman chemistry. Crude peptides are liberated from trifluoroacetic acid-labile linkers during the first cycle of Edman degradation and subsequently "embedded" in membranes. For peptides up to 20 residues, embedded sequencing repetitive yields were comparable to those of solid-phase sequencing. Preview sequencing of resin-bound Fmoc-synthesized peptides proved to be advantageous compared to other analytical methods, in that synthetic failures were detected and quantitated at the point of occurrence, regardless of whether incomplete Fmoc deprotection or incomplete coupling was responsible, and without interference from by-products formed during peptide-resin cleavage. Quantitative ninhydrin analysis, which previously has been found to give false positive results due to removal of the Fmoc group by a combination of reagents and high temperature, gave false negative results in this study, most probably due to incomplete removal of the Fmoc group prior to coupling. Quantitative sequence analysis results were supported by high-performance liquid chromatographic, amino acid and electrospray mass spectrometric analyses of the crude and purified peptides.

Amino Acid Sequence↗

PubMed1996

Monitoring of in vitro and in vivo exposure to sulfur mustard by GC/MS determination of the N-terminal valine adduct in hemoglobin after a modified Edman degradation.

We report that exposure to the chemical warfare agent sulfur mustard can be monitored by means of a modified Edman degradation involving selective release of the N-terminal valine adduct of hemoglobin with the agent. The degree of alkylation of the N-terminal valine in human hemoglobin is approximately 1-2% of the total alkylation induced in hemoglobin upon treatment of human blood with sulfur mustard. After modified Edman degradation, followed by derivatization with heptafluorobutyric anhydride, the obtained pentafluorophenyl thiohydantion derivative of the valine adduct could be analyzed at a > or = 0.5 fmol level by means of GC/MS under negative ion chemical ionization conditions. Applying this procedure, in vitro exposure of human blood to > or = 0.1 microM of sulfur mustard could be determined. In vivo exposure of guinea pigs could also be established at 48 h after intoxication intravenously with 0.5 mg/kg (0.06 LD50) of the agent.

Alkylation↗

PubMed1993

Amino acid sequence of a major human amelogenin protein employing Edman degradation and cDNA sequencing.

The abundant hydrophobic, proline-glutamine, and histidine-rich (over 90%) amelogenins constitute the major class of proteins in forming extracellular enamel matrix. These are thought to play a major role in the structural organization and mineralization of developing enamel. The present report describes the successful sequencing of the major human amelogenin protein, by use of both Edman degradation and cDNA sequencing. When Edman degradation was used, over 75% of the primary structure of the protein was determined. This sequence was supplemented with cDNA sequencing studies, which revealed the predicted sequence of this protein. Together, they provide the complete sequence of an important human enamel protein. The information complements recent studies on bovine and human amelogenin genes. A comparison between the present results and the protein sequences predicted from the corresponding human amelogenin genomic coding regions and that of cDNA sequences of other species is described.

Amelogenin↗

PubMedDate not supplied

Procedure for monitoring exposure to sulfur mustard based on modified edman degradation of globin.

A procedure for the modified Edman degradation of globin for determination of sulfur mustard adducts to the N-terminal valine residue in human hemoglobin has been developed for use under field laboratory conditions. The minimum detectable exposure level of human blood (in vitro) to sulfur mustard using this procedure is 100 nM. The interindividual and intraindividual variabilities of the procedure were acceptable (standard deviation < 10% and < 20%, respectively). The procedure could be properly set up and carried out in another laboratory within one working day, demonstrating its robustness.

Chemical Warfare Agents↗

PubMed1976

An improved procedure for automated Edman degradation used for determination of the N-terminal amino acid sequence of human transcobalamin I and human intrinsic factor.

An improved procedure for automated Edman degradation is presented. Three programs are described, one with double cleavage and two with single cleavage. The programs presented are characterized by a reversed delivery scheme for buffer and phenyl isothiocyanate, and by reduced cleavage times. The modified procedures applied on automated Edman degradation of the vitamin B12-binding proteins human transcobalamin I and human intrinsic factor, containing approximately 390 and 350 amino residues respectively, gave the following N-terminal amino acid sequences: Human transcobalamin I Glu-Ile-Cys-Glu-Val-Ser-Glu-Glu-Asn-Tyr-Ile-Arg-Leu-Lys-Pro-Leu-Leu-Asn-Thr-Met-Ile-Gln-Ser-Asn-Tyr-Asn-?-Gly- Human intrinsic factor Ser-Thr-Gln-Thr-Gln-Ser-Ser-Cys-Ser-Val-Pro-Ser-Ala-Gln-Glu-Pro-Leu-Val-Asn-Gly-Ile-Gln-?-Leu-Met-Glu-Thr- The background accumulation seems to be related not only to the length of the polypeptide chain being degraded, but also to the content of serine (and possibly threonine). A possible N leads to O acyl shift during the cleavage is a tentative explanation. The programs here represented lead to a significant reduction in background compared to conventional programs and allowed considerable prolongation of the degradations.

Amino Acid Sequence↗

PubMed1988

Sensitization of amino acid derivatives obtained from Edman degradation with radioactively-labeled iodohistamine.

The minimum amount of proteins and peptides required for sequencing is constantly decreasing as more sensitive microsequencing methods are developed. The sensitization of and Edman degradation product is one such method. We took the 2-anilino 5-thiazolinone amino acid intermediates obtained from Edman degradation by conventional sequencing procedures, and quantitatively reacted them with a primary amine. The amine used was radioactive [125I]iodohistamine, which affords highly sensitive detection. The labeled amino acid derivatives were separated by thin layer chromatography. Ten femtomoles of a labeled derivative can be detected by autoradiography.

Amino Acids↗

PubMed1985

An amino-terminal tryptophan derivative which is refractory to Edman degradation.

A new derivative of tryptophan is proposed to account for the observation that some peptides having amino-terminal tryptophan residues become refractory to Edman degradation. An acid-catalyzed oxidation of the indole nucleus and subsequent cyclization to a unique 3-anilinopyrrolidin-2-one derivative with cleavage of the peptide chain is the most likely chemical explanation. This amino acid is reactive with ninhydrin and contains an aryl amine. However, the amine does not bond to the alpha carbon so while reactive to phenyl isothiocyanate, distances are too great for the residue to be cleaved from the peptide during Edman degradation.

Amino Acid Sequence↗

PubMed1984

Use of o-phthalaldehyde to reduce background during automated Edman degradation.

Background generated during the cleavage step of the Edman degradation is a major obstacle to extended automated amino acid sequencing. It was demonstrated recently by A. S. Bhown, J. C. Bennett, P. H. Morgan, and J. E. Mole (1981, Anal. Biochem. 112, 158-162) that introduction of fluorescamine into the spinning cup reduced background by blocking primary amino groups at cycles where a proline residue is at the exposed amino terminus. A convenient blocking reaction program using o-phthalaldehyde which can be intercalated into the sequencing format of an automated Beckman liquid-phase sequencer is presented. The advantages of o-phthalaldehyde in blocking of primary amines when proline is amino terminal arise from its stability in aqueous solution and the ease of programmed metering of delivery to the sequencer spinning cup. The blocking reaction proved successful not only in extending sequence analyses but also in the elimination of unwanted sequences in selected peptide mixtures without the necessity of purification of the target peptide.

Aldehydes↗

PubMed1973

Edman degradation on in vitro biosynthesized peptidoglycans from Staphylococcus epidermidis.

Edman degradations were performed on the pentapeptide bridges of peptidoglycans which were biosynthesized in vitro by using six different species of seryl-transfer ribonucleic acid (tRNA). The ratio of glycine to serine in each of the five bridge positions varied with the concentration of crude glycyl-tRNA contained in the reaction mixtures, and serine appeared to be incorporated in a random manner. No single species of seryl-tRNA could be identified as the one active in in vivo pentapeptide bridge synthesis; however, it can be speculated that seryl-tRNA(s) from category I could be involved in the in vivo nonrandom incorporation of serine in the bridge.

Amino Acid Sequence↗

PubMed1980

Sequencing of peptide mixtures by Edman degradation and field-desorption mass spectrometry.

A new procedure is described for sequencing of peptide mixtures by a combination of Edman degradation and field-desorption mass spectrometry. The procedure involves measurement of the mass values of quasi-molecular ions ([M + H]+) of constituent peptides in the mixture and their fragments degraded by the Edman method. Calculation of all the possible mass differences of the mass values before and after degradation and identification of the phenylthiohydantoins released reveal the N-terminal amino acids of individual peptides in the mixture. Repetition of these operations gives the amino acid sequences of individual peptides in the mixture. As typical examples, the sequencing of peptide mixtures prepared by proteolytic digestion of glucagon are described. In addition, a computer program was designed for determining the possible sequences of proteins from the partial sequences and mass values of their proteolytic peptides. Output data showed that the entire amino acid sequence of glucagon can be determined by only four and two cycles of degradation of chymotryptic and tryptic peptides, respectively.

Amino Acid Sequence↗

PubMed1997

A role for Edman degradation in proteome studies.

Advances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two-dimensional electrophoresis. However, Edman degradation remains the principal technique for protein identification and it presents a significant bottleneck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman degradation to rapidly generate the powerful protein attribute, an N-terminal sequence tag.

Amino Acid Sequence↗