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PubMed1998

Sequence of canine COL1A2 cDNA: nucleotide substitutions affecting the cyanogen bromide peptide map of the alpha 2(I) chain.

The alpha2 chain of canine type I collagen was characterized with both sequence analysis of COL1A2 cDNA and chemical analysis of alpha2(I) chains. The complete sequence of canine COL1A2 cDNA was determined from reverse-transcribed and polymerase chain reaction-amplified total RNA from cultured skin fibroblasts. Pepsin-digested and cyanogen bromide-digested type I collagen peptides were analyzed with chromatography, gel electrophoresis, and mass spectrometry. Identity between the sequences of canine and human COL1A2 cDNA was 90.9%, predicting conservation of the 3 cysteine residues required for C-propeptide registration and of cleavage sites for signal peptidase, procollagen N-proteinase, vertebrate collagenase, and procollagen C-proteinase. Conservation of all 50 lysine residues was also predicted, including preservation of the 1:2 asymmetry in the X:Y distribution of the 31 lysine residues in the alpha2(I) triple helix. The human and canine sequences differed in the location of Y-position proline residues and the presence of two unique canine cyanogen bromide peptides, alpha2 CB3b and alpha2 CB3c,5. Knowledge of the conserved and unique features of canine COL1A2 will be valuable for analysis of the expression, synthesis, and structure of type I collagen as well as studies of canine osteogenesis imperfecta.

Amino Acid Sequence↗
PubMed2005

Fast on-column protein digestion with subsequent peptide mapping using tandem mass spectrometry with information dependent acquisition.

A platform for rapid on-line protein digestion of protein mixtures for direct infusion to a mass spectrometer is presented. A mixture of protein A, staphylococcal enterotoxin B and cytochrome c was used as a model mixture injected on a gel filtration column and a trypsin reactor which were connected in series to a micro liquid chromatography (microLC) system. The peptides in the column eluate were analyzed with ESI tandem mass spectrometry, utilizing information dependent acquisition (IDA). In one step, the proteins in the mixture (microM concentrations) were concomitantly desalted, separated, digested and identified with an overall analysis time of less than 40 min. Protein sequence coverage of 78-95% for the involved substances was achieved.

Amino Acid Sequence↗
PubMed2005

Identification of neutralizing epitopes on the VP2 protein of infectious bursal disease virus by phage-displayed heptapeptide library screening and synthetic peptide mapping.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease, which is one of the most important and widespread infectious diseases in commercial chickens. Conformational epitopes have been reported in the highly variable region of the VP2 protein of IBDV. In the present study, a random heptapeptide library was screened by using monoclonal antibodies (mAbs), YNW17 and YNW29, directed to the VP2 of IBDV and two peptide motifs, D-X-P-R and A-R-G, were identified. The motifs are present on the N and C terminal sequences of the highly variable region of VP2. Synthetic overlapping peptides covering the motifs on VP2 were analyzed by Dot- ELISA with the mAbs and two epitopes 197CDSSDRPRVYTIT209 and 329ARGSLAVTI337 identified. The above epitopes were also recognized by chicken anti-IBDV sera and shown to inhibit the binding of their mAbs to recombinant VP2. Both mAbs and sera from mice immunized with the conjugated epitope-peptides were able to neutralize serotype I IBDV. These results indicated that the epitopes are two neutralizing linear B-cell epitopes and would be useful for the development of peptide-based IBD vaccines.

Amino Acid Sequence↗
PubMed2002

Investigation of cytolysin variants by peptide mapping: enhanced protein characterization using complementary ionization and mass spectrometric techniques.

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have been used in conjunction with time-of-flight (TOF) and quadrupole ion trap (IT) mass spectrometry, respectively, to analyze various cytolysin proteins isolated from the sea anemone Stichodactyla helianthus and digested by the protease trypsin. By employing different ionization methods, the subsequent changes in ionization selectivity for the peptides in the digested protein samples resulted in ion abundance variation reflected in the mass spectra. Upon investigation of this variation generated by the two ionization processes, it has been shown in this study that enhanced protein coverage (e.g., >95% for cytolysin III) can be achieved. Additionally, capillary and microbore reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ESI mass spectrometry (MS) as well as flow injection analysis by nanoflow ESI-MS afforded the necessary limit of detection (LOD) for detailed structural information of the cytolysin proteins by tandem mass spectrometry (MS/MS) methods. It can be concluded that cytolysins II and III correspond to sticholysins I and II, that "cytolysin I" is a mixture of modified forms of cytolysins II and III, and that "cytolysin IV" is an incompletely processed precursor of cytolysin III.

Amino Acid Sequence↗
PubMed1987

Comparison of rat MHC class I antigens by peptide mapping.

Monoclonal antibodies specific for the rat major histocompatibility complex (MHC) class I antigens RT1.An, RT1.Au, and RT1.Eu were used for immunoprecipitation of antigens biosynthetically radiolabeled with 14C- or 3H-labeled arginine, lysine, and tyrosine; with arginine or tyrosine alone; and with or without tunicamycin in the culture medium. Heavy chains of the glycosylated and unglycosylated antigens were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their tryptic and chymotryptic peptides were compared by high performance liquid chromatography. The antigens coded by the same locus in two different haplotypes (An and Au) differed by 30%, whereas the products of two different loci in the same haplotype (Au and Eu) differed only by 1-3%. Comparative analysis of the data for samples labeled with single amino acids indicated that two amino acids in Au have been substituted by an arginine and probably by a tyrosine residue, respectively, in Eu. The high degree of homology between the products of the A and E loci in the same haplotype accounts for the difficulty in detecting recombinational events within the MHC of the rat by classical serological approaches.

Amino Acid Sequence↗
PubMed1997

ATP-dependent ferric hydroxamate transport system in Escherichia coli: periplasmic FhuD interacts with a periplasmic and with a transmembrane/cytoplasmic region of the integral membrane protein FhuB, as revealed by competitive peptide mapping.

The Escherichia coli iron transport system via ferrichrome belongs to the group of ATP-dependent transporters that are widely distributed in prokaryotes and eukaryotes. Transport across the cytoplasmic membrane is mediated by three proteins: FhuD in the periplasm, FhuB in the cytoplasmic membrane and FhuC (ATPase) associated with the inside of the cytoplasmic membrane. Interaction of FhuD with FhuB was studied in vitro with biotinylated synthetic 10 residue and 20-24 residue peptides of FhuB by determining the activity of beta-galactosidase linked to the peptides via streptavidin. Peptides identical in sequence to only one of the four periplasmic loops (loop 2), predicted by a transmembrane model of FhuB, and peptides representing a transmembrane segment and part of the adjacent cytoplasmic loop 7 of FhuB bound to FhuD. Decapeptides were transferred into the periplasm of cells through a FhuA deletion derivative that forms permanently open channels three times as large as the porins in the outer membrane. FhuB peptides that bound to FhuD inhibited ferrichrome transport, while peptides that did not bind to FhuD did not affect transport. These data led us to propose that the periplasmic FhuD interacts with a transmembrane region and the cytoplasmic segment 7 of FhuB. The transmembrane region may be part of a pore through which a portion of FhuD inserts into the cytoplasmic membrane during transport. The cytoplasmic segment 7 of FhuB contains the conserved amino acid sequence EAA...G (in FhuB DTA ...G) found in ABC transporters, which is predicted to interact with the cytoplasmic FhuC ATPase. Triggering of ATP hydrolysis by substrate-loaded FhuD may occur by physical interaction between FhuD and FhuC, which bind close to each other on loop 7. Although FhuB consists of two homologous halves, FhuB(N) and FhuB(C), the sites identified for FhuD-mediated ferrichrome transport are asymmetrically arranged.

Adenosine Triphosphate↗
PubMed2006

[Peptide mapping of the monoclonal antibodies against the heavy chain hemagglutinin from influenza virus H3N2].

Interaction of the synthetic peptides corresponding to the regions 122-133, 136-147, 154-164 and 314-328 of the virus A/Aichi/2/68 hemagglutinin heavy chain with monoclonal antibodies specific for this hemagglutinin was assayed in a variety of tests, e.g., ELISA, competition RIA, hemagglutinin-inhibition and virus-neutralization assays. The monoclonal antibody 152 reacted with the area 136-147 (epitope A), three monoclonal antibodies 3, 19 and 63 reacted exclusively with the area (154-164) Glu (epitope B). Mapping of two monoclonal antibodies IV A1 and IV G6 specific for the influenza virus A/Dunedin/ 4/73 hemagglutinin heavy chain and cross-reacting with a number of the H3 subtype viruses was carried out. The specificity of the interaction of the conservative peptide H3(314-328) with monoclonal antibodies IV A1 and IV G6 was confirmed by competition RIA and by competition hemagglutinin inhibition and virus neutralization.

Animals↗
PubMed1989

Peptide mapping and internal sequencing of proteins electroblotted from two-dimensional gels onto polyvinylidene difluoride membranes. A chromatographic procedure for separating proteins from detergents.

Direct sequence analysis of proteins electroblotted from two-dimensional polyacrylamide gels onto immobilizing matrices provides an efficient technique for obtaining N-terminal sequence data for proteins not amenable to purification by reversed-phase high-performance liquid chromatography (RP-HPLC). We present in this paper a procedure for obtaining peptide fragments from electroblotted proteins for internal amino acid sequence analysis. First, Coomassie Blue-stained proteins are extracted from polydivinylidene difluoride membranes, using a detergent mixture of sodium dodecylsulfate and Triton X-100. Proteins are then separated from the detergent mixture by a chromatographic procedure which relies on the ability of proteins to interact with certain reversed-phase sorbents at high organic solvent concentrations. Under these conditions, detergents and Coomassie Blue are not retained and pass through the column. Proteins are recovered by simultaneously: (i) introducing trifluoroacetic acid into the mobile phase and (ii) decreasing the organic solvent concentration. After proteolytic fragmentation, peptides are purified by microbore-column (1-2 mm I.D.) RP-HPLC for microsequence analysis.

Amino Acid Sequence↗
PubMed1997

Candidate reference methods for hemoglobin A1c based on peptide mapping.

A reference method that specifically measures hemoglobin (Hb) A1c is an essential part of the reference system for the international standardization of Hb A1c/glycohemoglobin. We have developed a new method for quantification, based on the specific N-terminal residue of the hemoglobin beta-chains. Enzymatic cleavage of the intact hemoglobin molecule with endoproteinase Glu-C has been optimized to obtain the beta-N-terminal hexapeptides of Hb A1c and Hb A0. These peptides have been separated by reversed-phase HPLC and quantitated by electrospray ionization-mass spectrometry (method A) or by capillary electrophoresis (method B). With these peptides and hyphenated separation techniques, it has been possible to overcome the insufficient resolution of currently used protein separation systems for Hb A1c.

Chromatography, High Pressure Liquid↗
PubMed1994

Matrix-assisted laser desorption mass spectrometric peptide mapping of proteins separated by two-dimensional gel electrophoresis: determination of phosphorylation in synapsin I.

A technique is described for the rapid, sensitive analysis of posttranslational modifications of proteins that have been separated by 2-dimensional electrophoresis and blotted onto a membrane with a cationic surface. The isolated protein spots visualized by reverse staining of the blotting membrane are excised, washed, and subjected to chemical (cyanogen bromide) and/or enzymatic (endoproteinase Lys-C) degradation directly on the membrane. The resulting mixture of peptide fragments is extracted from the membrane into a solution that is compatible with matrix-assisted laser desorption mass spectrometric analysis and analyzed without fractionation. Relatively accurate (+/- 1 Da) mass determination of these peptide fragments provides a facile and sensitive means for detecting the presence of modifications and for correlating such modifications with the differential mobility of different isoforms of a given protein during 2-dimensional electrophoresis. The technique is applied to the determination of sites of phosphorylation in synapsins Ia and Ib, neuronal phosphoproteins that are believed to function in the regulation of neurotransmitter release and are substrates for cAMP and Ca2+/calmodulin-dependent protein kinases, which appear to control their biological activity.

Amino Acid Sequence↗
PubMed1988

Single-step purification of two hyperglycaemic neurohormones from the sinus gland of Procambarus bouvieri. Comparative peptide mapping by means of high-performance liquid chromatography.

A crude aqueous extract from 2000 sinus glands of the Mexican crayfish Procambarus bouvieri (Ortmann) was fractionated on a muBondapak-Phenyl column. Two isoforms of the crustacean hyperglycaemic hormone, designated CHH-B and CHH-C in order of elution, were isolated in pure form. Their biochemical characterization showed a remarkable degree of homology. A tryptic digest of each isohormone was fractionated on an Ultrasphere-ODS column. Only one tryptic peptide in CHH-C was eluted later than its homologous peptide in CHH-B. On acid hydrolysis both tryptic peptides had the same composition but, as they contain Asp and Glu, we suspect that the difference residues in a double reciprocal amidation-deamidation of two acidic residues.

Amino Acids↗
PubMed2004

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping.

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.

Alkanesulfonates↗