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At least 397 records · Page 22Linked to original sources
PubMed1997

Separation and characterization of the tryptic peptide mapping of recombinant bovine growth hormone by reversed-phase high-performance liquid chromatography electrospray mass spectrometry.

Reversed phase high-performance liquid chromatography (RPHPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) was used to separate and characterize the peptides resulting from the tryptic cleavage of somidobove, a recombinant bovine growth hormone. The tryptic digestion of somidobove was carried out at room temperature for 15 hours. The tryptic peptides were separated on a Zorbax SB300 C8 column with trifluoroacetic acid--acetonitrile gradient elution and characterized by LC/ESI-MS. Resulting single and combined peptide fragments were characterized by comparing the observed molecular mass with the calculated mass for these fragments.

Amino Acid Sequence↗
PubMed1997

Peptide mapping and amino acid sequencing of two catechol 1,2-dioxygenases (CD I1 and CD I2) from Acinetobacter lwoffii K24.

The partial amino acid sequences of two catechol 1,2-dioxygenases (CD I1 and CD I2) from Acinetobacter lwoffii K24 have been determined by analysis of peptides after cleavages with endopeptidase Lys-C, endopeptidase Glu-C, trypsin, and chemicals (cyanogen bromide and BNPS-skatole). They include 248 amino acid sequences (4 fragments) of CD I1 and 211 amino acid sequences (5 fragments) of CD I2. Two enzymes have more than 50% sequence homology with type I catechol 1,2-dioxygenases and less than 30% sequence homology with type II catechol 1,2-dioxygenases. Two enzymes have similar hydropathy profiles in the N-terminal region, suggesting that they have similar secondary structures.

Acinetobacter↗
PubMed1991

Peptide mapping of different Bacillus thuringiensis toxin gene products by CNBr cleavage in SDS-PAGE gels.

A cyanogen bromide fragmentation reaction of 3 hr in sodium dodecyl sulfate gel slices was used for the degradation of the toxins coded by the three cryIA genes from Bacillus thuringiensis kurstaki. Peptide patterns diagnostic for each toxin gene product were observed. Treatment longer than 3 hr led to the weakening and disappearance of protein bands. In 9 out of 16 wild B. thuringiensis strains tested, it was determined that only one of the cryIA genes was being expressed; in 6 strains, one to two genes were identified, with the presence of an additional gene possible and masked because of the overlap of peptide bands; in one strain none of the genes was expressed.

Bacillus thuringiensis↗
PubMed1988

Structure and function of the sigma-70 subunit of Escherichia coli RNA polymerase. Monoclonal antibodies: localization of epitopes by peptide mapping and effects on transcription.

Murine monoclonal antibodies reactive with the major sigma subunit (sigma-70) of Escherichia coli RNA polymerase were obtained by standard hybridoma techniques. Western blot analyses established that seven antibodies had unique specificities after various chemical and enzymatic methods were used to fragment sigma. Peptides were purified by HPLC using size-exclusion, reverse-phase, or ion-exchange chromatography. The epitopes for six of these antibodies have been localized to specific peptides. These peptides were further characterized by amino acid composition and N-terminal sequencing. Sigma, which has a molecular weight of 70.2K, runs as 83K on SDS gels in this study. This anomalous behavior has been localized to the very acidic N-terminal half of the molecule. One antibody is unable to bind to native sigma. Two others do not bind well to sigma when it is contained in holoenzyme, indicating that their epitopes are in regions of sigma which are inaccessible in the holoenzyme complex. All three of these antibodies fail to inhibit in vitro transcription by holoenzyme. The other four antibodies all can inhibit in vitro transcription.

Antibodies, Monoclonal↗
PubMed1989

Suppression effects in peptide mapping by plasma desorption mass spectrometry.

Suppression effects observed in plasma desorption (PD) mass spectrometry have been studied and compared to those found in fast atom bombardment mass spectrometry. A basic difference in the mechanism of suppression in the two techniques is demonstrated. In positive ion mode PD mass spectrometry, peptides carrying net positive charges are preferentially detected when analysed together with peptides carrying net negative charges, and in negative ion mode PD mass spectrometry, the situation is generally reversed. Based on this complementarity, PD mapping carried out by enzymatic digestion of a nitrocellulose-bound sample can in many cases yield almost complete coverage of smaller proteins.

Animals↗
PubMed1991

Identification of the site of interaction between cytochrome c3 and ferredoxin using peptide mapping of the cross-linked complex.

Structural studies carried out on a cross-linked complex between cytochrome c3 and ferredoxin I, both isolated from Desulfovibrio desulfuricans Norway, allowed the identification of the site of interaction between the two redox proteins. Staphylococcus aureus proteinase and chymotrypsin digestions led to characterization of peptides containing both cytochrome c3 and ferredoxin sequences. The cytochrome c3 sequences involved in the three isolated cross-linked peptides contained several lysine residues localized around the heme 4 crevice. This analysis stressed the peculiar role of lysines 100, 101, 103, 104 and 113, which could be considered as major cross-link sites, as opposed to the lysines 75, 79 and 82, which could be considered as minor cross-link sites. One cross-linked peptide, containing two ferredoxin sequences joined to one cytochrome c3 sequence, had been isolated, suggesting the possibility of more than one cross-link per covalent complex. All these results led to the identification of heme 4 of cytochrome c3 as the site of interaction for the ferredoxin I. This study confirms the proposal that could be deduced from the hypothetical structure of the complex built by computer graphics modelling (Cambillau, C., Frey, M., Mosse, J., Guerlesquin, F. and Bruschi, M. (1988) Proteins: struct., funct. genet. 4, 63-70).

Binding Sites↗
PubMed1994

Peptide mapping using combinations of size-exclusion chromatography, reversed-phase chromatography and capillary electrophoresis.

Recombinant extracellular superoxide dismutase was proteolytically degraded by trypsin and the digest was thereafter separated using three different separation techniques. The size differences of the obtained fragments were exploited by size-exclusion chromatography (SEC) on a highly efficient column for peptide separation. Collected fractions representing different size groups of peptides were then separated on the basis of hydrophobicity on reversed-phase liquid chromatography (RPLC). This led to simplified RPLC-chromatograms where collected peaks were considerably more pure than if the digest was separated direct on RPLC and the identity of eluted peaks could easily be determined by amino acid analysis. Finally the digest was run on capillary electrophoresis for separation based on charge differences. For the identification of the different peaks in the electropherogram, the total digest was spiked with known peptides purified by the two other separation techniques. This technique proved to be very powerful for peak identification in an electropherogram from a digest composed of many fragments eluting closely together.

Amino Acid Sequence↗
PubMed2004

Peptide mapping of feline immunodeficiency virus by IFN-gamma ELISPOT.

Interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) has become an important tool in studying antigen-specific T lymphocyte responses. Soluble peptides can be used to map T-cell epitopes, providing information that is useful in the design and evaluation of vaccines as well as studies of immunopathogenesis. To date, this assay has not been widely utilized in feline immunodeficiency virus (FIV) research. We have developed a feline IFN-gamma ELISPOT assay and used it to determine FIV-specific T-cell epitopes recognized by infected cats. A panel of 331 peptides, 15 amino acids in length and overlapping by 10 residues was synthesized. The peptide library spanned the FIV structural (Gag), envelope (Env), reverse transcriptase (RT), and open-reading-frame A (OrfA) proteins. Initially, 34 pools, containing 7-10 peptides each were screened by IFN-gamma ELISPOT against peripheral blood mononuclear cells (PBMC) from eight cats chronically infected with the NCSU(1) molecular clone of FIV and four uninfected control cats. Individual peptides from pools recognized by FIV+ cats were then evaluated and optimal peptides were combined into pools representing Gag, Env, RT, and OrfA. A higher percentage of FIV infected cats were identified as responders against the peptide pools when using fresh PBMC as compared to cryopreserved PBMC. In vitro restimulation of cryopreserved PBMC with the peptide pools improved the sensitivity of the assay to similar levels as observed from fresh samples. Individual peptides used in the pools were generally found to stimulate CD8+ T-cells more efficiently than CD4+ T-cells. Comparison of the peptide sequences to representative FIV sequences from clades A-D showed conservation was high among Gag and RT peptides, variable among Env peptides and low for OrfA peptides. The IFN-gamma ELISPOT assay and FIV-specific peptide pools we describe here will be useful in assessing cell-mediated responses to experimental FIV vaccines.

Amino Acid Sequence↗