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PubMedDate not supplied

Routine sensitive peptide mapping using LC/MS of therapeutic proteins produced by recombinant DNA technology.

At the Sixth International Conference on Methods in Protein Sequence Analysis held in Seattle, Washington, August 17-21, 1986, Frank Robey of the Food and Drug Administration (FDA) proposed a "wish list" of possible future techniques for the analysis of recombinant products. Included in this list was liquid chromatography (HPLC) in-line with mass spectrometry for peptide mapping. This wish has come true. In 1988, a new technique, electrospray ionization (ESI) of liquids with quadrupole mass analysis, became a major breakthrough in accurately measuring molecular weights of proteins up to 100,000 daltons or more. Now, using an affordable, simple, easy-to-use single-stage quadrupole mass spectrometer, peptide mapping is routine.

Amino Acid Sequence↗
PubMed1993

Peptide mapping reveals differences in the non-glycosylated domains of cystic fibrosis and normal tracheobronchial mucins.

Tracheobronchial mucins from lung mucus secretions of healthy individuals and from patients with cystic fibrosis (CF) were purified according to a protocol established in our laboratory. Following digestion of the purified, reduced-alkylated mucin (free of 118 kDa and 70 kDa components) with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2 and TR-3) were observed upon chromatography on a Superose 6 column using FPLC. TR-1 (glycosylated fraction) contained all of the carbohydrate, while TR-2 and TR-3 fractions had no detectable sugars. Comparison of the amino acid composition of TR-1 fractions from normal and CF individuals revealed no significant differences, while the TR-2 fractions from these mucins showed noticeable differences. Peptide mapping of TR-2 fractions from normal and CF mucins was performed on a C18 reverse phase column using FPLC. The peptide maps of normal mucins were markedly different from CF mucins. A greater number of peptides were seen in the TR-2 fractions of normal mucins when compared to CF mucin TR-2 fractions. In addition, normal TR-2 fractions appeared to be comprised of more hydrophobic peptides when compared to CF TR-2 fractions. These data provide evidence of possible structural differences in the non-glycosylated regions of CF and non-CF mucins, since the TR-2 fractions are essentially derived from the T-domains in the "naked" stretches of the mucin polypeptide backbone.

Amino Acids↗
PubMed1996

Human neurotrophin-3: a one-step peptide mapping method and complete disulfide characterization of the recombinant protein.

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an initial denaturation of NT-3 in 6 M guanidinium chloride (pH6) for 2 hr at 37 degrees C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110. This disulfide structure is homologous to the NGF family of neurotrophic factors.

Amino Acid Sequence↗
PubMed1988

Peptide mapping on Northern blot analyses of insulin receptors in brain and adipocytes.

Previous studies have demonstrated differences in the size of insulin receptor subunits in brain and adipocytes that appear to involve variations in glycosylation of the proteins. In this report, we examined the degree of homology in the protein backbones of insulin receptors in both tissues by peptide mapping and compared the mRNAs encoding the receptors by Northern blot analysis. Photoaffinity-labeled insulin receptors from rat brain and adipocytes were deglycosylated and then subjected to partial proteolysis by five different enzymes with differing substrate specificities. The intact receptors and their proteolytic fragments were analyzed by electrophoresis and autoradiography. Each enzyme yielded a unique pattern of fragments ranging from 70 to 11 kDa. In all cases, there was a striking similarity in the peptide maps generated from insulin receptors in brain and adipocytes. Northern hybridization experiments were carried out using poly(A)+ RNA from rat brain, rat adipocytes, and human hepatocarcinoma (HEP G2) cells. In rat brain, two bands of 9.5 and 7.4 kb were detected and, in rat adipocytes, the same two bands were observed. The two mRNA bands observed in rat tissues represented only two of the five mRNA species seen in human HEP G2 cells. The results indicate that the protein domains and the mRNAs encoding of insulin receptors in brain and adipocytes are very similar, if not identical.

Adipose Tissue↗
PubMed1986

Characterization of rabbit uterine estrogen receptor proteins by radioiodination and partial peptide mapping.

Two estrogen binding proteins (Mr = 50,000 and 65,000) were purified from rabbit uterine cytosol using an improved procedure for affinity chromatography on diethylstilbestrol-agarose. The estrogen receptors were radioiodinated while adsorbed to the resin using the lactoperoxidase or Bolton-Hunter techniques. After elution, the labeled receptors were utilized for peptide mapping studies and investigations of receptor function. Partial peptide mapping revealed strong homology between the Mr 50,000 and 65,000 proteins suggesting common structural features. Estrogen receptors labeled by the lactoperoxidase procedure were rendered unable to bind immobilized heparin or hormone; in contrast, the Bolton-Hunter labeling technique yields proteins that retain both their ability to bind hormone and to absorb on heparin-agarose. The development of these iodination methodologies appears useful for the investigation of both the structure and functional properties of the receptor proteins.

Animals↗
PubMed1993

RP-HPLC peptide mapping methods for the analysis of recombinant human pro-urokinase.

Among the techniques available for the detection of protein structure variants such as single point mutations, RP-HPLC peptide mapping plays a key role owing to the high reproducibility of peptide retention times, determined as identity indexes. Because of the possible co-elution of some proteolytic fragments, an improvement of the array of information given by the technique can be achieved by setting up a series of experiments under hydrolytic conditions with different enzymes, followed by appropriate RP-HPLC gradient elutions. Such an experimental approach appears to be particularly useful in the examination of proteins with a high molecular weight, where the resulting RP-HPLC maps are complex. Therefore different RP-HPLC peptide mapping methods have been studied for recombinant human pro-urokinase (r-h-proUK), a thrombolytic agent of apparent molecular weight of 46 kD. The RP-HPLC maps indicate that the methods developed are not only suitable for the qualitative control of the amino acid sequence and arrangement of disulphide bonds but also represent the first demonstration of the identity of the primary structure of the recombinant and of the native species, within the limits of the technique.

Amino Acid Sequence↗
PubMed1984

Peptide maps and N-terminal sequences of polypeptides from early region 1A of human adenovirus 5.

Experiments exploring the reasons for a multiplicity of products from early region 1A of adenovirus 5 are described. Labeled early region 1A products from wild-type virus were synthesized in infected cells and in a cell-free system programmed with mRNA from infected cells, immunoprecipitated specifically with an antipeptide serum, E1A-C1, directed against the C-terminal sequence of E1A products, and separated by gel electrophoresis. Two-dimensional maps of [35S]methionine-labeled peptides were consistent with antigens of 52,000 daltons (52K) and 48.5K being from the 13S mRNA and antigens of 50K, 45K, and 35K from the 12S mRNA. Partial N-terminal sequences of 52K, 50K, 48.5K, and 45K synthesized in vitro showed that each of these antigens was initiated at the predicted ATG at nucleotide 560 in the DNA sequence. These results eliminate multiple initiation sites and proteolytic cleavage at the N-terminal end as sources of antigen diversity. Peptide maps and N-terminal sequences were obtained in a similar way for E1A products from the Ad5 deletion mutant dl1504, which lacks the normal initiator codon. As predicted, these polypeptides are initiated at the next ATG, 15 codons downstream in the wild-type sequence. These results are discussed in relation to Kozak's ribosomal scanning model.

Adenoviruses, Human↗
PubMed1982

Peptide mapping of fat cell membrane substrates for cholera toxin-catalyzed ADP-ribosylation.

Incubating rat fat cell membranes with [32P]NAD+ and cholera toxin results in ADP-ribosylation of three distinct components with approximate molecular weights of 42,000, 46,000 and 48,000. Partial proteolytic peptide maps of the Mr = 46,000 and 48,000 toxin-specific substrates generated by elastase, alpha-chymotrypsin, or Staphylococcus aureus V-8 protease were nearly identical, while those of the Mr = 42,000 target lacked several peptides common to both of the larger molecular weight targets. In addition, peptide maps generated from the Mr = 42,000 target displayed a number of peptides which were absent from the maps generated from either the Mr = 46,000 or 48,000 targets. These data suggest that the Mr = 46,000 and 48,000 substrates are closely related proteins, however the relationship between the Mr = 42,000 toxin-specific substrate and the larger peptides remains to be established. The relative patterns of fat cell membrane labelling by cholera toxin in the presence of [32P]NAD+ were identical in hypothyroid as compared to euthyroid rat fat cells.

Adenosine Diphosphate Ribose↗
PubMed2000

Peptide mapping and characterisation of glycation patterns of the glima 38 antigen recognised by autoantibodies in Type I diabetic patients.

AIMS/HYPOTHESIS: Glima 38 is an N-glycated neuroendocrine membrane protein of M(r) 38,000, which is recognised by autoantibodies in approximately 20% of patients with Type I (insulin-dependent) diabetes mellitus. The aim of this study was to characterise the carbohydrate moiety and generate peptide maps of glima 38. METHODS: Sera of high immunoreactivity to glima 38 were used to isolate 35-S methionine-labelled protein from betaTC-3 cells and a neuronal cell line GT1.7. Tunicamycin was used to inhibit N-glycation of glima 38 and define the core protein. The carbohydrate moiety was characterised for tunicamycin sensitivity, lectin binding and susceptibility to different endoglycosidases. The protein moiety was subjected to digestion by proteases to define peptide maps. RESULTS: The autoreactive epitopes in glima 38 recognised by Type I diabetic sera are conformational and independent of the carbohydrate moiety. Inhibition of N-glycation of glima 38 in vivo, shows a protein core of M(r) 22,000 in both pancreatic beta-(betaTC3) and neuronal (GT1.7) cell lines. The carbohydrate moieties in the two cell types are distinct but contain a similar amount of terminal sialic acid residues and at least five oligosaccharide chains Glima 38 binds Triticum vulgare and Ricinus communis I lectins. Endoproteinase treatment of the M(r) 22,000 core protein results in peptides of M(r) 4500 and M(r) 20,000 with Lys-C, and peptides of M(r) 4000 and M(r) 11,000-12,000 with Glu-C/V8 and Asp-N proteases. CONCLUSION/INTERPRETATION: The biochemical properties of glima 38 define it as a new autoantigen in Type I diabetes and provide a basis for its purification.

Adolescent↗
PubMed2000

Improvements in protein identification by MALDI-TOF-MS peptide mapping.

A new strategy for identifying proteins in sequence data-bases by MALDI-MS peptide mapping is reported. The strategy corrects for systematic deviations of determined peptide molecular masses using information contained in the opened database and thereby renders unnecessary internal spectrum calibration. As a result, data acquisition is simplified and less error prone. Performance of the new strategy is demonstrated by identification of a set of recombinant, human cDNA expression products as well as native proteins isolated from crude mouse brain extracts by 2-D electrophoresis. Using one set of calibration constants for the mass spectrometric analyses, 20 proteins were identified without applying any molecular weight restrictions, which was not possible without data correction. A sequence database search program has been written that performs all necessary calculations automatically, access to which will be provided to the scientific community in the Internet.

Animals↗
PubMed1982

A sensitive peptide mapping method. Identification of three amino acid substitutions within two anti-azobenzenearsonate monoclonal antibody light chains.

Peptide mixtures, precolumn-derivatized with dimethylaminoazobenzene isothiocyanate, have been separated by reversed-phase high-performance liquid chromatography to generate a dimethylaminoazobenzene thiocarbamoyl peptide map. The eluted peptide derivatives are detected in the visible region with a sensitivity of 2-5 pmol and can be collected for direct structural analysis. This technique was applied to compare the sequence homology of two immunoglobulin light chains which were derived from two anti-azobenzenearsonate monclonal antibodies, namely 10K44-7A1 and 10K26-12A1. The complete variable region sequences of 10K44-7A1 and 10K26-12A1 light chains were established based on the sequence analysis of tryptic peptides, intact light chains and reference sequences obtained previously [Siegelman M. and Capra, J.D. (1981) Proc. Natl Acad. Sci. USA, 78, 7679-7683]. Altogether, three amino acid substitutions have been detected within complementary determining regions 1 and 2, and framework region 3, all requiring only a single base change at the DNA level. This new technique provides detection limits and the feasibility of analysing peptides which are not obtainable with conventional techniques.

Amino Acid Sequence↗
PubMedDate not supplied

Application of the multiple antigenic peptides (MAP) strategy to the production of prohormone convertases antibodies: synthesis, characterization and use of 8-branched immunogenic peptides.

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediately following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84-99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, 1H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventional method of peptide-carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the case of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.

Amino Acid Sequence↗
PubMed1982

Peptide maps of regulatory subunits of Bacillus subtilis RNA polymerase.

Peptide maps of four regulatory subunits of Bacillus subtilis RNA polymerase were obtained. Three sigma-like proteins (sigma 55, sigma 37, and sigma 29) as well as the transcription modification factor, delta (delta) protein, were shown to give unique peptide patterns. This observation demonstrates that each is a distinct protein species; none is derived from another by a simple proteolytic modification.

Bacillus subtilis↗
PubMed1982

Peptide mapping of 125I-labelled membrane protein of influenza viruses by reverse-phase high-performance liquid chromatography.

The resolution potential of reverse-phase high-performance liquid chromatography (HPLC) for peptide analysis of hydrophobic viral membranes has been investigated, using as model the membrane (M) protein of influenza virus. Proteolytic digests of 125I-labelled M protein and CNBr fragments, extracted from radioiodinated whole virus, have been separated on a uBondapak C18 column with an isopropanol or acetonitrile solvent system. Peptide mapping of trypsin digests of M protein from A/PR/8/34 (H1N1) and A/chicken/Germany/N/49 (H10N7) viruses was identical, whereas Staphylococcus aureus V8 protease digests showed minor differences in at least two peptides. The results also show that HPLC is a powerful tool for the separation of proteolytic digests of viral proteins, since the peptide maps are highly reproducible and recovery was greater than 85%.

Chromatography, High Pressure Liquid↗
PubMed1995

Photoaffinity-labeled ligand binding domains on dopamine transporters identified by peptide mapping.

Binding domains on rat dopamine transporters for cocaine and 1-(2-diphenylmethoxy)ethyl-4-(3-phenylpropyl)piperazine compounds were identified using controlled proteolysis of photoaffinity-labeled protein and epitope-specific immunoprecipitation of the labeled fragments. Rat dopamine transporters were photoaffinity labeled with 1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido- 3-[125I]iodophenyl)ethyl]piperazine ([125I]DEEP) [a 1-(2-di- phenylmethoxy)ethyl-4-(3-phenylpropyl)piperazine analog] or 3 beta-(p-chlorophenyl)tropane-2 beta-carboxylic acid, 4'-azido-3'- [125I]iodophenylethyl ester ([125I]RTI 82) (a cocaine analog) and were gel purified to remove contaminating radioactivity. The resulting samples were treated with V8 protease or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide maps generated with each enzyme were different for each of the ligands, suggesting that the ligands were incorporated into different regions of the protein. Identical peptide maps were generated from striatum- and nucleus accumbens-derived transporters, indicating that these polypeptides are highly similar in primary sequence. The proteolytic fragments generated by V8 protease were localized to specific domains of the protein using antipeptide antibodies corresponding to five different regions of the transporter. Fragments of 10 and 7 kDa from [125I]DEEP-labeled transporters were specifically immunoprecipitated with an antibody generated against amino acids 42-59 (near the first putative trans-membrane domain), whereas a 34-kDa fragment from [125I]RTI 82-labeled transporters was precipitated with three different sera corresponding to regions in the carboxyl-terminal two thirds of the protein. None of the V8 fragments smaller than 45 kDa, containing either photolabel, was altered in molecular mass by N-deglycosylation. The results indicate that photoincorporation of [125I]DEEP occurs in the amino half of the dopamine transporter, near the first two transmembrane helices, whereas [125I]RTI 82 labels the carboxyl-terminal region of the protein, between transmembrane domains 4 and 12.

Affinity Labels↗
PubMed1999

Variant antibody identification by peptide mapping.

During development of CGP56901, a monoclonal antibody (MAb) specific for a unique epitope on human IgE, the protein A-purified IgG from one of the candidate production cell lines, showed an additional minor heavy chain (H-chain) band with a molecular weight slightly lower than that of the principal H-chain band on SDS-PAGE. The N-terminal amino acid sequence of this minor H-chain species indicated that at least the first 30 amino acids were identical to those of the antibody light-chain (L-chain) variable domain. More detailed studies using peptide mapping and amino acid sequencing analysis confirmed a crossover event between the V genes of the antibody. The position is between Arg108 of the L chain and Ala124 of the H chain. This crossover resulted in a variant H chain, which had 16 fewer amino acid residues than the normal CGP56901 H chain. These results show that peptide mapping is a useful "first-line" analytical tool in the characterization of the quality of the monoclonal antibody.

Amino Acid Sequence↗
PubMed1984

Evidence from peptide mapping for a human renin zymogen.

Data obtained from peptide mapping of the active and inactive forms of human renin show that there are extensive regions of common sequence in the two forms of the enzyme, and are consistent with the hypothesis that inactive renin is a renin zymogen.

Chromatography, High Pressure Liquid↗