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At least 217 records · Page 12Linked to original sources
PubMed1994

Amino acid sequence of a porcine zona pellucida glycoprotein ZP4 determined by peptide mapping and cDNA cloning.

Mammalian zona pellucida is an attractive target for developing a contraceptive vaccine. In this study, the amino acid sequence of a core protein of porcine zona pellucida glycoprotein ZP4 was determined by peptide mapping and cDNA cloning. Two kinds of ZP4 peptides were identified: one consisted of 128 amino acid residues and the other of 133 amino acid residues with an additional five amino acid sequence at the carboxy-terminal end of the 128 amino acid peptide. Both peptides had two potential N-linked glycosylation sites. The 128 amino acid peptide showed 39.1% similarity to the amino-terminal region of mouse ZP2 polypeptide. The positions of five cysteine residues were the same for porcine ZP4 and mouse ZP2. The cloned cDNA possessed an additional 195 nucleotides at the 3' end of the sequence corresponding to the 133 amino acid peptide. This additional sequence was found to encode the amino-terminal 10 amino acid sequence of porcine ZP2 polypeptide. These results suggest that porcine ZP4 and ZP2 are derived from a common parent polypeptide by proteolytic cleavage at the position between 133 and 134 residues.

Amino Acid Sequence↗
PubMedDate not supplied

Peptide mapping characterization by capillary electrophoresis of a human monoclonal anti-Rh(D) antibody produced for clinical study.

CE has been employed for peptide mapping characterization of the light chain of a human anti-Rhesus (D) (Rh[D]) antibody presently undergoing clinical evaluation. In the presence of an ion-pairing agent used to increase resolution, reproducible maps were obtained within 55 min after injection of 12 fmol of protein. CE has also been employed as a direct and quick screening tool for purity evaluation of the tryptic peptides obtained from reversed-phase high-performance liquid chromatography (RP-HPLC) prior to sequence analysis. Post-translational modifications of the protein were determined by identification of the cysteine residues implicated in disulfide linkages.

Amino Acid Sequence↗
PubMed1979

Analytical peptide mapping by high performance liquid chromatography. Application to intestinal calcium-binding proteins.

Peptide mapping of underivatized tryptic digests of bovine and chick intestinal calcium-binding proteins has been accomplished by high performance liquid chromatography (HPLC). High precision analysis of nanomolar quantities of peptides were achieved in less than 1 h (recycle time). Peak resolution and definition are superior compared to conventional techniques and recoveries of both small (4-residue) hydrophilic and large (30-residue) hydrophobic peptides are excellent. The total amino acid composition of the bovine intestinal calcium-binding protein has been accounted for on the basis of two tryptic maps of 20 microgram of protein each.

Amino Acids↗
PubMed1979

Subunit structure and peptide mapping of junctional and extrajunctional acetylcholine receptors from rat muscle.

We have purified the junctional acetylcholine receptor from normal rat skeletal muscle and compared its structure with that of the extrajunctional receptor from denervated muscle. The two receptors from leg muscle were distinguished by isoelectric focusing and by reaction with sera from patients with myasthenia gravis. The junctional form of the acetylcholine receptor was purified from normal leg muscle by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose followed by sucrose gradient centrifugation. Analysis of radioiodinated receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the subunit structure of the junctional receptor was similar to that previously determined for the extra-junctional form (Froehner, S. C., et al. (1977) J. Biol. Chem. 252, 8589-8596), with major polypeptides, whose apparent molecular weights in 9% polyacrylamide gels were 45 000 and 51 000. In addition, several minor polypeptides were found. When the two receptors were labeled with different isotopes of iodine and run together on a sodium dodecyl sulfate gel, the subunits of one receptor could not be resolved from those of the other. As seen earlier with the extrajunctional form, the affinity alkylating reagent [3H]MBTA labeled the 45 000- and 49 000-dalton polypeptides of the junctional receptor. Peptide mapping showed that the two MBTA binding subunits are structurally related, although they are unrelated to the other polypeptides, and that the 45 000- and 51 000-dalton polypeptides of the junctional receptor were indistinguishable from those of the extrajunctional receptor. In addition, peptide mapping of the four subunits of acetylcholine receptor isolated from Torpedo californica electric organ showed that these four polypeptides appear to be structurally unrelated.

Acetylcholine↗
PubMed1993

Structural analysis of tissue inhibitor of metalloproteinases-1 (TIMP-1) by tryptic peptide mapping.

Tryptic digests of recombinant TIMP-1 have been resolved on reverse-phase HPLC and the major peaks identified by N-terminal sequencing. This procedure accounted for the entire molecule, except two short peptides of 2 and 4 amino acids in length. The peptide map was used to (i), characterize an insoluble 'core' peptide seen on digestion of TIMP-1 in non-reducing conditions; (ii), confirm the structure of delta 127-184TIMP-1, a recently described truncated form of the TIMP-1 molecule; (iii), identify exposed regions of the intact and truncated TIMP-1 molecules by measuring the rate of tryptic peptide release and (iv), locate sites of aberrant proteolysis seen when recombinant human TIMP-1 was purified at large scale.

Amino Acid Sequence↗
PubMed1993

Peptide mapping using EOF-driven capillary isoelectric focusing.

Capillary isoelectric focusing (CIEF) in uncoated and commercially available coated capillaries was applied to the separation of tryptic peptides from bovine and chicken cytochrome c. This is the first report of peptide mapping by CIEF. Before separation, the pIs of individual peptides expected to be formed during the digest were calculated, and then the separation pattern obtained was correlated with the calculated pIs. This correlation was reasonably good for some peptides, but not as good for others. However, differentiation of the two species digests was easily achieved based on the CIEF map. The major limitation of the method is the fact that uv detection at 280 nm must be employed, meaning that only tryptophan and tyrosine containing peptides will be detected, since the ampholytes used to generate the pH gradient absorb below 280 nm. Post-column derivatization of the peptides is one solution to this problem. Migration time reproducibility was on the order of 2%, and run times were less than 20 min for the entire pH gradient range in the uncoated capillary. In the coated capillary, superior resolution was obtained at the expense of longer run times.

Amino Acid Sequence↗
PubMed2003

Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization-time of flight-mass spectrometry.

Peptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH(4)HF(2). The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 50 degrees C, even 20 fmol cytochrome c could be well digested and detected.

Adsorption↗
PubMed1992

Chromogranin A epitopes: clues from synthetic peptides and peptide mapping.

Chromogranin A (CgA) is a 48 kDa acidic protein in neuroendocrine secretory vesicles whose primary structure is now known. We used synthetic peptides, synthetic peptide antisera, intact molecule antisera, chymotryptic peptide mapping, microsequencing, immunoblotting, and immunoprecipitation to probe the location of immunodominant domains within the CgA molecule. Polyclonal anti mid-molecule, anti N-terminal and anti C-terminal antibodies specifically visualized CgA (both bovine and human) in one and two dimensional immunoblots of adrenal chromaffin vesicles, and the stain CgA fragments further suggested bidirectional (both N- and C-terminal) cleavage or processing of CgA. Anti intact CgA immunoblotting of HPLC-separated peptides from chymotrypsin-digested bovine CgA revealed several strongly immunoreactive internal peptides, two of which were positioned by N-terminal amino acid sequencing: CgA91ff. and CgA197ff.. A single synthetic peptide (CgA79-113) was recognized by three antibodies developed against the intact CgA molecule: two polyclonal rabbit antisera as well as a monoclonal mouse antibody. Not all antigenicity algorithm-predicted domains were immunogenic, suggesting that some of these predicted domains may not be accessible. Polyclonal anti mid-molecule, anti N- and anti C-terminal synthetic peptide antisera specifically immunoprecipitated 125I-labeled bovine CgA from aqueous solution; mid-molecule antisera precipitated substantially greater amounts than terminal antisera. The immunoprecipitation results suggested exposed terminal as well as interior hydrophilic epitopes in the molecule in its intact, native conformation. 125I-human CgA was best precipitated by anti N-terminal antisera, consistent with greatest interspecies sequence conservation at the N-terminus of CgA. The terminal antisera reacted immunohistochemically in a granular pattern with adrenal medullary chromaffin cells (but not adrenal cortical cells) and pancreatic islet cells (but not pancreatic exocrine acini). Thus, synthetic and chymotryptic peptides yielded novel and specific insights into the structure, conformation, vesicular processing and interior immunodominant domains of CgA.

Amino Acid Sequence↗
PubMed2004

An improved model for prediction of retention times of tryptic peptides in ion pair reversed-phase HPLC: its application to protein peptide mapping by off-line HPLC-MALDI MS.

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-A pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.

Amino Acid Sequence↗
PubMed1988

Comparison of two different benzodiazepine binding proteins by peptide mapping after limited proteolysis.

Proteins P51 and P55 were photolabeled by [3H]flunitrazepam, [3H]clonazepam or 3H-Ro 15-4513 and then compared by peptide mapping after limited digestion with various proteases. Results indicated that [3H]flunitrazepam or [3H]clonazepam irreversibly bind to the same and [3H]Ro 15-4513 to a different part of these proteins. The different radiolabeled peptide patterns obtained from P51 or P55, irrespective of the photolabel used, suggest a difference in the molecular structure of these proteins.

Animals↗
PubMedDate not supplied

Fish species identification by peptide mapping of the myosin heavy chain.

Fish species of seafood made of washed fish flesh (e.g. imitation crab meat from surimi) were identified by peptide mapping of the myosin heavy chain (MHC). In the first step the MHC was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5% T gel, followed by a second SDS-PAGE in a 15% T gel with proteolytic digestion of the MHC in the gel. The resulting peptides gave species specific patterns.

Animals↗
PubMed2000

An on-line protein digestion device for rapid peptide mapping by electrospray mass spectrometry.

A simple laboratory-constructed device has been developed for fast on-line protein digestion followed by peptide mapping by use of electrospray mass spectrometry. Taking advantage of its nonsolubility properties at near-neutral pH values, pepsin could be nonpermanently attached to the PEEK tube commonly employed as transfer capillary between the syringe and the electrospray ion source. After optimization of experimental conditions such as pH, solvent, and exposure time, efficient digestion of several model proteins of molecular weights between 14,000 and 66,000, some having disulfide bridges, was successfully carried out. This technique provided reliable and reproducible sequence information by means of C-terminal-specific cleavages of aromatic and hydrophobic residues. As an application, protein identification could be achieved using a protein database search software.

Amino Acid Sequence↗
PubMed1994

Bovine pancreatic deoxyribonuclease F: isoelectric focusing, peptide mapping and primary structure.

DNAase F is a minor isoform of bovine pancreatic DNAase which can be separated from the other isoforms (DNAases A, B, C and D) present in a commercial preparation by a preparative isoelectric-focusing cell (Rotofor; Bio-Rad). The ampholytes and other contaminating proteins present in DNAase F preparations can be removed by chromatography on an affinity column (Cibacron Blue 3GA-agarose) and a hydrophobic-interaction column (phenyl-Sepharose CL-4B). The complete separation of DNAase F from the other isoforms is demonstrated on a thin-layer isoelectric-focusing gel, DNAase F being the most basic (pI 5.68). A procedure is described for tryptic peptide mapping by h.p.l.c. requiring only picomolar amounts of DNAase F protein. The DNAase F map shows two peptide peaks not present in the DNAase A map, and the DNAase F map does not have a peak at the position where a C-terminal peptide of DNAase A is normally eluted. The amino acid compositions and sequences for the two new peptides suggest that Gly240 in DNAase A is changed to Arg240 in DNAase F.

Amino Acid Sequence↗
PubMed2005

Artificial neural network modeling of peptide mobility and peptide mapping in capillary zone electrophoresis.

Recently, we have developed an artificial neural network model, which was able to predict accurately the electrophoretic mobilities of relatively small peptides. To examine the robustness of this methodology, a 3-3-1 back-propagation artificial neural network (BP-ANN) model was developed using the same inputs as the previous model, which were the Offord's charge over mass term (Q/M(2/3)), corrected steric substituent constant (E(s,c)) and molar refractivity (MR). The data set consisted of 102 peptides with a larger range of size than that of our earlier report - up to 42 amino acid residues as compared to 13 amino acids in the initial study - that also included highly charged and hydrophobic peptides. The entire data set was obtained from the published result by Janini and co-workers. The results of this model are compared with those obtained using multiple linear regressions (MLR) model developed in this work and the multi-variable model released by Janini et al. Better predictive ability of the BP-ANN model over the MLR indicates the non-linear characteristics of the electrophoretic mobility of peptides. The present model exhibits better robustness than the MLR models in predicting CZE mobilities of a diverse data set at different experimental conditions. To explore the utility of the ANN model in simulation of the CZE peptide maps, the profiles for the endoproteinase digests of melittin, glucagon and horse cytochrome C is studied in the present work.

Amino Acid Sequence↗
PubMed1995

Tryptic peptide mapping of sequence 299-585 of human serum albumin by high-performance liquid chromatography and fast atom bombardment mass spectrometry.

The determination of the tryptic peptide mapping of sequence 299-585 (cyanogen bromide fragment A) of human serum albumin (HSA) by chemical and enzymatic cleavages and combined use of HPLC and FAB-MS is described. Reduction and carboxymethylation of A gave four subfragments which were separated by HPLC and digested with trypsin. Tryptic fragments were separated by HPLC and identified by FAB-MS. A total coverage of about 95% of the entire sequence was obtained. Tryptic fragments not identified include mostly single amino acids and very hydrophilic peptides which were absent in the chromatograms. The high reproducibility of the experiments and the satisfactory yield of the tryptic fragments identified demonstrate the great potential of the combined use of HPLC separation and FAB-MS analysis for the structural investigation of HSA.

Amino Acid Sequence↗
PubMed1994

Tryptic peptide mapping of sequence 1-298 of human serum albumin by high-performance liquid chromatography and fast-atom bombardment mass spectrometry.

The determination of the tryptic peptide mapping of sequence 1-298 of human serum albumin (HSA) by chemical and enzymatic cleavage and combined use of high-performance liquid chromatography (HPLC) and fast-atom bombardment mass spectrometry (FAB-MS) is described. A total coverage of about 75% of the entire sequence was obtained. Unidentified fragments included some peptides which were not present in the chromatograms because of their extreme hydrophobic or hydrophilic character, as indicated by the calculated retention times. Due to the high reproducibility of the experiments and to the satisfactory yield of the tryptic fragments identified, the combined use of HPLC and FAB-MS appears to possess great potential for structural investigation of HSA.

Amino Acid Sequence↗
PubMed2004

A simple tool for drawing proteolytic peptide maps.

UNLABELLED: I describe a simple standalone program that assists in the preparation of peptide digestion maps. These are useful for comparative studies and for locating peptides within a primary sequence. The program creates an output file as scalable vector graphics that can then be viewed in a web browser or imported into a graphics-editing program. AVAILABILITY: The program, as a standalone executable, is available upon request from the author. CONTACT: r.beynon@liv.ac.uk.

Computer Graphics↗
PubMed1993

Localisation of a receptor-recognition domain on the S3 subunit of pertussis toxin by peptide mapping.

Overlapping 10-amino-acid peptides, which consecutively span the amino acid sequence of the S3 subunit of pertussis toxin, were synthesised on polyethylene pins and screened for their ability to bind the glycoprotein fetuin. Fetuin binding was localised to a single peptide comprising amino acids 46-55. A free peptide, (E)S3c, of longer sequence (S3 amino acids 44-58) was also found to bind alpha-1-acid glycoprotein, mixed brain gangliosides and fetuin. (E)S3c also recognised asialofetuin but with a lower apparent affinity relative to fetuin. The single tryptophan residue of the peptide yielded a fluorescence-emission maximum of 355 nm. In the presence of either ganglioside or the phospholipid L-alpha-lysolecithin, but not N-acetylneuramin-lactose or lactosylceramide, the emission intensity of (E)S3c was enhanced and the emission maximum blue-shifted to 340 nm by ganglioside, or to 345 nm by L-alpha-lysolecithin. Monosialogangliosides, disialogangliosides, and trisialogangliosides, when fluorescence-titrated, were each found to bind the peptide with a similar dissociation constant of 4.4 +/- 2.8 microM. These findings demonstrate that region 44-58 of the pertussis-toxin S3 subunit is likely to be involved in the recognition of both glycosylated and phospholipid constituents of target-cell membranes.

Amino Acid Sequence↗