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PubMed1996

Improved peptide mapping using phytic acid as ion-pairing buffer additive in capillary electrophoresis.

By digestion of the highly basic polypeptide aprotinin or bovine pancreatic trypsin inhibitor (BPTI) with endoproteinase Lys-C after unfolding, reduction and pyridylethylation, five fragments are obtained. These fragments are separated by free solution capillary electrophoresis using a phosphate buffer at neutral pH. The effect of the ion-pairing buffer additive phytic acid on the separation was investigated. It is shown that phytic acid through ion-pair formation influences the mobility of only those peptide fragments having a net positive charge at the pH of the separation buffer. The affinity of phytic acid to the peptides correlates with their isoelectric point and the charge to mass ratios. Hence, by changing the concentration of phytic acid, it is possible to manipulate the migration order and the separation of the peptides.

Amino Acid Sequence↗
PubMed1989

Peptide mapping of conformational epitopes in a human malarial parasite heat shock protein.

A protein of 75 kDa is found in large quantities throughout the blood stages of the human malarial parasite, Plasmodium falciparum. Based on a partial amino acid sequence for p75, previously deduced from a cDNA clone encoding approximately 40% of the molecule, secondary structural predictions were made. The potential role of long range effects on the tertiary structure of the protein stabilized by disulfide bridges was determined by reduction and alkylation of the fusion protein. Five regions were then chosen for peptide modeling. Peptides of 16, 28, 49, 64, and 76 residues were synthesized and used to immunize rabbits. All but the 16-residue peptides were capable of stimulating boostable IgG antibody responses in rabbits, but the antibody produced against the 49 mer did not react with the native parasite protein. Thus, the 28, 64, and 76 residue peptides represent good immunologic models for portions of the P. falciparum 75-kDa protein capable of stimulating both T and B cells in rabbits. The peptides were also used to probe whether any of the selected regions contain epitopes which react with antibodies from owl monkeys immune to P. falciparum. Of these peptides, two were found to be consistently recognized in ELISA by four owl monkey antisera raised in response to malarial infection. Because these two peptides model a cysteine-containing region of the protein, owl monkey sera were also used as probes of the importance of disulfide bonding in maintaining the native structure. The results obtained were consistent with a folding pattern for p75 that incorporates a disulfide bond between cysteines 161 and 194. These results also suggest that most of the epitopes recognized in this part of p75 by the immune system of the monkey are created by folding of the molecule.

Amino Acid Sequence↗
PubMed1986

Interspecies conservation of structure of interphotoreceptor retinoid-binding protein. Similarities and differences as adjudged by peptide mapping and N-terminal sequencing.

Structural properties of the retinal extracellular-matrix glycolipoprotein interphotoreceptor retinoid-binding protein (IRBP) from human, monkey and bovine retinas have been compared. SDS/polyacrylamide-gel-electrophoretic analysis of limited tryptic and Staphylococcus aureus-V8-proteinase digests show virtually identical patterns for the monkey and human proteins, whereas both sets differ considerably from the bovine protein pattern. Time-course digestion shows monkey IRBP to be more readily cleaved than bovine IRBP and also cleaved to smaller fragments. Also, reversed-phase h.p.l.c. of complete tryptic digests of the IRBPs indicate that, although they have in common a similar preponderance of hydrophobic peptides, all three proteins differ extensively in their fine structure. The N-terminal sequences of monkey and bovine IRBPs have been extended beyond those presented in our previous report [Redmond, Wiggert, Robey, Nguyen, Lewis, Lee & Chader (1985) Biochemistry 24, 787-793] to over 30 residues each. The sequences yet show extensive homology, differing at only two positions, although the major monkey sequence has an additional five amino acid residues at its N-terminus ('n + 5' sequence) not observed with bovine IRBP ('n' sequence). The newly determined N-terminal sequence of human IRBP demonstrates the presence of equal amounts of the 'n' and 'n+5' sequences that are qualitatively identical with those of the monkey. The presence of the five-amino-acid-residue extension in primate, but not bovine, IRBP may indicate variation in post-translational processing.

Amino Acid Sequence↗
PubMed1995

Microscale immobilized protease reactor columns for peptide mapping by liquid chromatography/mass spectral analyses.

Methods and procedures for the construction and operation of a microscale immobilized protease (trypsin) reactor are described. Optimization of reactor efficiency with regard to reactor flowrate, length, and temperature as well as sample concentration has been examined, and a novel system for sample preconcentration is presented. The structural analysis of a standard protein (cytochrome c) by a combination of trypsin microreactor digestion followed by off-line electrospray ionization MS analysis is demonstrated at the 10-pmol level. The application of these techniques for the rapid confirmation (1-2 h) of a genetic variant (hemoglobin E) including MS/MS analysis of the variant peptide is included.

Amino Acid Sequence↗
PubMed1988

Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping.

1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells.

Binding Sites↗
PubMed1998

Differences and similarities between Trichinella spiralis and T. pseudospiralis in morphology of stichocyte granules, peptide maps of excretory and secretory (E-S) products and messenger RNA of stichosomal glycoproteins.

The ultrastructure of muscle larvae of Trichinella pseudospiralis was studied by electron microscopy. The overall structure of muscle larvae of T. pseudospiralis resembled that of T. spiralis except for the stichocyte granules. T. pseudospiralis had at least 3 kinds of stichocyte granules distinguishable from each other by their shape, size and inclusions. The granules had some resemblance to alpha granules or beta granules of T. spiralis, but no resemblance to gamma granules. In favour of these morphological differences and similarities among T. spiralis and T. pseudospiralis, excretory and secretory (E-S) products (originating from stichocyte granules) of the 2 species differed to some degree. In an analysis by 2-dimensional electrophoresis, some peptide spots migrating at 45 kDa were shared by the 2 species but the other spots were unique to each of the 2 species. Messenger RNA encoding the 43 kDa glycoprotein of stichocyte granules was detected in the muscle larvae of both species but mRNA encoding the 53 kDa glycoprotein was detected only in muscle larvae of T. spiralis.

Animals↗
PubMed1991

Peptide mapping through the coupling of capillary electrophoresis and high-performance liquid chromatography: map prediction of the tryptic digest of myoglobin.

The tryptic map of horse myoglobin was analysed through capillary electrophoresis using capillaries modified by a monolayer of acrylamide. The results were reproducible and the map was obtained in less than 30 min from ca. 8 pmol of tryptic digest. The peptide identification was performed using peptides previously identified by high-performance liquid chromatography. The peak areas measured using the two techniques are closely related, and the comparison of elution and migration times shows that the two techniques provide different maps. Furthermore, using the semiempirical relationship suggested by Grossman et al. [Anal. Biochem., 179 (1989) 28], which links the electrophoretic mobility to the charge of the peptide and its number of amino acids, a good agreement between predicted and experimental mobilities was observed.

Amino Acids↗
PubMed1997

Peptide mapping of the [125I]Iodoazidoketanserin and [125I]2-N-[(3'-iodo-4'-azidophenyl)propionyl]tetrabenazine binding sites for the synaptic vesicle monoamine transporter.

The full-length cDNA for the rat recombinant synaptic vesicle monoamine transporter (rVMAT2) containing a COOH-terminal polyhistidine epitope was engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed to high levels and purified to >95% homogeneity using immobilized Ni2+-affinity chromatography followed by lectin (concanavalin A) chromatography. Purified transporter was photolabeled using [125I]-7-azido-8-iodoketanserin ([125I]AZIK) and [125I]2-N-[(3'-iodo-4'-azidophenyl)propionyl]tetrabenazine ([125I]TBZ-AIPP). Both [125I]AZIK and [125I]TBZ-AIPP photoaffinity labeling of purified rVMAT2 were protectable by 10 microM tetrabenazine (TBZ), 10 microM 7-aminoketanserin, and 1 mM concentrations of the transporter substrates dopamine, norepinephrine, and serotonin. Radiolabeled peptides were generated using enzymatic and chemical methods, purified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH2-terminal microsequenced. Radiosequencing of [125I]AZIK-labeled rVMAT2 indicated derivatization of Lys-20 in the NH2 terminus, just prior to putative transmembrane domain 1 (TMD1). [125I]TBZ-AIPP derivatized a segment of rVMAT2 between Gly-408 and Cys-431 in TMD10 and 11. These data implicate juxtaposition of TMD1 and 10/11.

Affinity Labels↗
PubMed1997

Identification of heparin-binding domains in the amyloid precursor protein of Alzheimer's disease by deletion mutagenesis and peptide mapping.

Recent studies have shown that the binding of the amyloid protein precursor (APP) of Alzheimer's disease to heparan sulfate proteoglycans (HSPGs) can modulate a neurite outgrowth-promoting function associated with APP. We used three different approaches to identify heparin-binding domains in APP. First, as heparin-binding domains are likely to be within highly folded regions of proteins, we analyzed the secondary structure of APP using several predictive algorithms. This analysis showed that two regions of APP695 contain a high degree of secondary structure, and clusters of basic residues, considered mandatory for heparin binding, were found, principally within these regions. To determine which domains of APP bind heparin, deletion mutants of APP695 were prepared and analyzed for binding to a heparin affinity column. The results suggested that there must be at least two distinct heparin-binding regions in APP. To identify novel heparin-binding regions, peptides homologous to candidate heparin-binding domains were analyzed for their ability to bind heparin. These experiments suggested that APP contains at least four heparin-binding domains. The presence of more than one heparin-binding domain on APP suggests the possibility that APP may interact with more than one type of glycosaminoglycan.

Alzheimer Disease↗
PubMed1994

Peptide mapping of subtilisins as a practical tool for locating protein sequence errors during extensive protein engineering projects.

Protein engineering based on rational design is an iterative process of sequential amino acid residue replacements. This requires a rapid and sensitive method for checking the protein sequence after each round of mutagenesis. As shown with subtilisin BL, acid treatment followed by urea denaturation renders the enzyme degradable by trypsin within 10 min. Separation of the peptides by reversed-phase HPLC produces a map that differentiates even the most conservative alteration on peptides as large as 48 amino acid residues. The method was used to uncover erroneous mutations; to determine the concentration of active protease relative to an internal standard of known specific activity; to measure the rate of oxidation of methionine-216 in the oxyanion hole of subtilisin BL; and to document that under these conditions no other methionine in the molecule is oxidized by hydrogen peroxide.

Amino Acid Sequence↗
PubMed1988

Peptide mapping studies of the pertussis toxin substrate in human neutrophils, platelets and erythrocytes.

Protease digestion of the ADP-ribosylated pertussis toxin substrate (PTS) protein was carried out after solubilization with SDS (Cleveland gels) and in the intact membrane. Cleveland gel analysis showed substantial similarities in the maps for the PTS component in neutrophils, platelets and erythrocytes and also in the S49 AC-lymphoma cell line. In the intact membrane ADP-ribosylation followed by digestion showed limited access of proteases to the PTS component. Of eight proteases tested, only papain and Staphylococcus aureus gave substantial digestion. This pattern was observed in the human platelet, erythrocyte and neutrophil plasma membranes. When the sequence was reversed and ADP-ribosylation was carried out after protease digestion, a very different pattern was observed with much greater susceptibility to digestion being noted with several proteases. By contrast, analysis of the murine AC-membrane showed some minor variations in the digest patterns. In addition, under all three conditions tested, maps of the cholera toxin substrate for the human platelet showed remarkable similarities to those obtained with the pertussis toxin substrate. Our results indicate that the protease sensitive sites of the alpha subunit of PTS and protection from proteolysis after ADP-ribosylation are properties which are shared by the PTS components of human platelets, erythrocytes and neutrophils.

Adenosine Diphosphate Ribose↗
PubMed2002

Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis.

Transthyretin (TTR) is a 127-amino acid residue transport protein. In plasma, TTR exists as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR are hypothesized to destabilize the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with extracellular deposition of wild-type TTR, its variants or fragments as amyloid fibrils in various tissues and organs. A definitive diagnosis of ATTR depends on the detection and identification of TTR variants. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), in combination with trypsin digestion, have been shown to be powerful tools in characterizing TTR variants. Typically, TTR or its tryptic digest is analyzed by MALDI-TOF MS, liquid chromatography ESI MS, or both. Analysis of tryptic digests by MALDI-TOF MS does not provide enough sequence coverage in TTR to identify all possible modifications. To improve sequence coverage, aliquots of immunoprecipitated TTR samples were digested with trypsin, lysyl endopeptidase Lys-C, or endoproteinase Asp-N. Identification of the peptides from each digest by MALDI-TOF MS provided preliminary information about the sites and mass shifts due to amino acid substitutions from genetic mutations and to posttranslational modifications. The location and identity of the modifications in the variant proteins were then confirmed by tandem mass spectrometry, accurate mass measurements, and direct DNA sequence analysis. Using these methodologies, we achieved 100% sequence coverage. The detection of two nonpathologic variants (Thr119Met and Gly6Ser) and four pathologic variants (Phe64Leu, Asp38Ala, Phe44Ser, and previously unreported Trp41Leu) are described as illustrations of this approach.

Adult↗
PubMed1990

Immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognized by human and woodchuck antibodies.

Hepatitis delta virus (HDV) is a defective RNA virus which is dependent on hepatitis B virus for essential helper functions. Only a single highly basic phosphoprotein, HDV antigen (HDAg), is expressed by the HDV genome during infection in humans. Antibody directed to HDAg is important in the diagnosis of HDV infection, and it is likely but not yet proven that the immune response to HDAg provides significant protection against subsequent exposures to HDV. In an effort to map the antigenic domains of HDAg, 209 overlapping hexapeptides, spanning the entire 214 amino acid residues of the protein, were synthesized on polyethylene pins and probed by enzyme-linked immunosorbent assay with sera containing high titers of anti-HD antibodies. Domains recognized by antibodies present in serum from human chronic carriers of this virus included residues 2 to 7, 63 to 74, 86 to 91, 94 to 100, 159 to 172, 174 to 195, and 197 to 207. Antibody from an acutely superinfected woodchuck recognized similar epitopes, as well as a domain spanning residues 121 to 128. Together, residues in these antigenic domains constitute 41% of the HDAg molecule. Oligopeptides 15 to 29 residues in length and representing epitopes of HDAg found to be dominant in humans (residues 2 to 17, 156 to 184, and 197 to 211) were synthesized in bulk and found to possess significant antigenic activity by microdilution enzyme-linked immunosorbent assay. The reactivity of peptide 197-211 with human sera confirms that the entire 214 amino acids of HDAg are expressed during infection in vivo. In addition, these results suggest that synthetic peptides may be useful reagents for development of new and improved diagnostic tests for HDV infection.

Amino Acid Sequence↗
PubMed1989

Comparative studies on microsomal NADPH-cytochrome P-450 reductase using a monoclonal antibody: tissue distribution, specific activity and peptide mapping.

1. In order to elucidate the molecular structure and the distribution of the enzyme in different microsomes, specific antibodies have been developed against rabbit liver NADPH-cytochrome P-450 reductase. 2. The monoclonal antibody (MAb B1) against rabbit liver reductase cross-reacted well with reductases from various animal species and those from various tissues of the rabbit. 3. NADPH-cytochrome P-450 reductase from rabbit tissues such as liver, lung, adrenal gland, kidney and polymorphonuclear leukocyte were closely related in structure and antigenic properties, in addition to having similar catalytic properties. 4. No multiple forms of the reductase in the rabbit were observed in liver nor in other tissues.

Animals↗
PubMed2002

Structural characterization of monomeric folding intermediates of recombinant human macrophage-colony stimulating factor beta (rhM-CSFbeta) by chemical trapping, chromatographic separation and mass spectrometric peptide mapping.

We have developed a strategy for the characterization of protein folding intermediates that combines selective modification of bis-cysteinyl thiol groups with melarsen oxide (MEL), chromatographic separation and mass spectrometric characterization of the resulting protein derivatives. In the unfolding reaction of recombinant human macrophage-colony stimulating-factor beta (rhM-CSFbeta) we observed monomeric M.4MEL and dimeric D.2MEL intermediates. The major locations of the MEL groups in D.2MEL were at C157 and C159. In M.4MEL, MEL groups were predominantly located at C31 and C102. These results indicate the presence of highly structured dimeric and monomeric intermediates. In the completely reduced R.4MEL derivative, MEL groups were distributed such that the smallest ring structures resulted.

Chromatography, Gel↗