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At least 379 records · Page 21Linked to original sources
PubMed1997

Prostate-specific antigen: characterization of epitopes by synthetic peptide mapping and inhibition studies.

To improve our understanding of the prostate-specific antigen (PSA) antigenic regions, we studied the association targets of one anti-PSA polyclonal antibody and 10 anti-PSA monoclonal antibodies (mAbs). We also examined the ability of the mAbs to inhibit PSA enzymatic activity and block the association of PSA with alpha 1-antichymotrypsin (ACT). Linear epitope mapping with a polyclonal antibody indicated the presence of six major antigenic regions in PSA. Examination of the panel of mAbs established that three of them bind to linear epitopes. Five of the mAbs inhibited > 90% of PSA enzymatic activity. However, inhibition of PSA enzymatic activity and hindrance of PSA-ACT association by mAbs cannot be used to predict whether the mAbs bind to free PSA, the PSA-ACT complex, or both. Some of the mAbs may block PSA-ACT association through peripheral occlusion of the binding site, or through induction of conformational changes in PSA.

Amino Acid Sequence↗
PubMed1990

Evidence by peptide mapping that the region CD4(81-92) is involved in gp120/CD4 interaction leading to HIV infection and HIV-induced syncytium formation.

Peptide fragments of the CD4 molecule were compared in their ability to 1) inhibit CD4-dependent HIV-induced cell fusion; 2) inhibit CD4-dependent HIV infection in vitro; and 3) block gp120 envelope glycoprotein binding to CD4. Peptides from the region CD4(81-92), although inactive when underivatized, were equipotent inhibitors of CD4-dependent virus infection, cell fusion, and CD4/gp120 binding when derivatized via benzylation and acetylation. Peptides of identical chemical composition, but altered sequence and derivatization pattern that blocked gp120 binding to either CD4-positive cells or solubilized CD4, also blocked infection and fusion with similar potencies. Those that did not block gp120/CD4 interaction were also inactive in HIV-1 infection and cell fusion assays. No other peptide fragments of the CD4 molecule inhibited fusion, infection, or CD4/gp120 interaction. The peptide CD4(23-56), derived from a region of CD4 implicated in binding of CD4 antibodies that neutralize HIV infection and cell fusion, had no effect on CD4-dependent cell fusion, HIV-1 infection, or CD4/gp120 binding, but did reverse OKT4A and anti-Leu 3a blockade of gp120 binding to CD4. These data provide evidence that the 81-92 region of CD4 is directly involved in gp120 binding leading to CD4-dependent HIV infection and syncytium formation. Previous observations with structural mutants of CD4 suggest that the CDR2-homologous region of CD4 is also involved, either directly or indirectly, in binding of gp120 to CD4. The CDR2- and CDR3-like domains of CD4 may both contribute to the binding of the HIV envelope necessary for HIV-1 infection and HIV-1-induced cell fusion.

Antibodies, Monoclonal↗
PubMed2002

Combined in-gel tryptic digestion and CNBr cleavage for the generation of peptide maps of an integral membrane protein with MALDI-TOF mass spectrometry.

A limitation of the in-gel approaches for the generation of peptides of membrane proteins is the size and hydrophobicity of the fragments generated. For membrane proteins like the lactose transporter (LacS) of Streptococcus thermophilus, tryptic digestion or CNBr cleavage yields several hydrophobic fragments larger than 3.5 kDa. As a result, the sequence coverage of the membrane domain is low when the in-gel tryptic-digested or CNBr-cleaved fragments are analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). The combination of tryptic digestion and subsequent CNBr cleavage on the same gel pieces containing LacS approximately doubled the coverage of the hydrophobic membrane domain compared to the individual cleavage methods, while the coverage of the soluble domain remained complete. The fragments formed are predominantly below m/z 2500, which allows accurate mass measurement.

Amino Acid Sequence↗
PubMed2003

Bacillus spore identification via proteolytic peptide mapping with a miniaturized MALDI TOF mass spectrometer.

An approach is tested here as a rapid screening method for Bacillus spore species employing bacterial peptide analysis with a miniaturized MALDI TOF mass spectrometer. A limited set of tryptic peptides was generated in situ following selective solubilization of the small, acid-soluble protein family (SASP) from spore samples on the MALDI sample holder. To facilitate species identification, a compact database was created comprising masses of the tryptic cleavage products generated in silico from all Bacillus and Clostridium SASPs whose sequences are available in public databases. Experimental measurements were matched against the custom-made database, and a published statistical model was then used to evaluate the probability of false identifications.

Bacillus↗
PubMed1992

Peptide mapping and microsequencing of proteins separated by SDS-PAGE after limited in situ acid hydrolysis.

A method is described for the isolation of peptide fragments from proteins separated by polyacrylamide gel electrophoresis. After completion of the electrophoresis step, gels are stained with Ponceau S or Coomassie Blue. Gel portions containing protein stained with Ponceau S are excised and transferred to borosilicate glass digestion tubes containing 0.9 ml of 1 mM NaOH or 5 mM Na2HPO4. After complete dissociation of the dye from the protein, 0.1 ml of 20% formic acid is added and the protein is hydrolyzed in situ at 112 degrees C for four hours. Subsequently the acid solution is made 10% in acetonitrile and chromatographed as such on a C18 (C4) reversed-phase column using an appropriate large-volume sample loading syringe and injection loop. Proteins stained with Coomassie Blue can be hydrolyzed in situ after complete removal of the dye with an aqueous solution containing 40% acetone, 10% triethylamine and 5% acetic acid. The gel slices are next washed with HPLC-grade water and protein is hydrolyzed in 2% formic acid under standard conditions. Gel-related contaminants do not interfere with the peptide separation under the proper conditions of HPLC analysis.

Amino Acid Sequence↗
PubMed1988

Identification of a Glu greater than Lys substitution in the activation segment of human pepsinogen A-3 and -5 isozymogens by peptide mapping using endoproteinase Lys-C.

The isozymogens PGA-3 and PGA-5 of human pepsinogen A were digested with endoproteinase Lys-C. The peptides were separated by reverse-phase HPLC. PGA-5 showed a peak strongly absorbing at 254 nm absent in PGA-3. Analysis of amino acid composition using the Pico-Tag methodology combined with DABITC-sequencing reveals the sequence Tyr-Phe-Pro-Gln-Trp-Lys (peptide 37-43 of the activation segment). This confirms a study at the DNA level by our group [16] suggesting a Glu greater than Lys mutation at position 43 in the activation segment of PGA-5. Furthermore, it is proposed that the number of genetic variants of PGA is higher than is actually seen by electrophoresis.

Amino Acid Sequence↗
PubMed1997

Hydrophobic peptide mapping of clinically relevant heptathelical membrane proteins by capillary electrophoresis.

The structural investigation of G protein-coupled receptors has been hindered by the lack of techniques to effectively resolve the hydrophobic peptides obtained by chemical or proteolytic cleavage, as well as the minute amounts of protein typically isolated. We have developed a capillary electrophoresis method for efficient separation of hydrophobic peptides using a cyanogen bromide digest of bacteriorhodopsin as a model for these clinically important membrane proteins. This procedure includes (i) solubilization of the protein digest in acetic acid; and (ii) electrophoresis using an acetic acid-based buffer system augmented by acetonitrile and hexane sulfonic acid, in a Polybrene-coated fused silica capillary. The potential for detection sensitivity to be increased at least 100-fold by use of on-line solid-phase extraction on C18-silica is shown. This approach is potentially useful for peptide fingerprinting of sparse and extremely hydrophobic membrane receptors.

Bacteriorhodopsins↗
PubMed1994

Peptide mapping of bovine and chicken cytochrome c by capillary isoelectric focusing with universal concentration gradient imaging.

Capillary isoelectric focusing with universal concentration gradient imaging detection was used to separate and detect tryptic peptides from bovine and chicken cytochrome c. For a desalted sample of peptide angiotensin 2, the isoelectric point (pI) measured by the instrument agreed well with the pI calculated from amino acid pK values. For the cytochrome digests, correlations between measured and calculated pI values were imprecise because peak positions shifted slightly from test to test. This problem is thought to be caused by the inefficient desalting process used on the samples, leaving salt residues which caused distortion in the pH gradient during the focusing process. However, this system differentiated between the two cytochrome c's. The concentration gradient imaging detected peptides which contain no tyrosine and no tryptophan amino acids, which a UV absorption detector operating at 280 nm could not. The separation and detection steps took only 5-7 min because no mobilization was necessary after the focusing process.

Amino Acid Sequence↗
PubMed1997

High-sensitivity peptide mapping by micro-LC with on-line membrane blotting and subsequent detection by scanning-IR-MALDI mass spectrometry.

A novel approach to the on-line mass determination of peptides from digested proteins by scanning infrared matrix-assisted laser desorption/ionization (scanning-IR-MALDI) is described. The peptides were continuously collected directly onto a PVDF (polyvinylidene fluoride) strip during a HPLC run. Individual peptides were detected by lining up the PVDF strip with the UV trace from the HPLC run, using visible dye markers as reference points. The local resolution of the peptides on the PVDF membrane is preserved during matrix incubation for MALDI-MS as shown by comparing the UV chromatogram and the total ion current (TIC) from an on-line coupled electrospray ionization (ESI) mass spectrometer with the scanning-IR-MALDI data from the corresponding areas on the PVDF strip. The intensities of the mass profiles obtained by scanning-IR-MALDI reflect the amount of peptides present on the PVDF strip. The higher sensitivity of IR-MALDI-MS yielded mass information not detectable by ESI-MS. After the scanning-IR-MALDI experiment, the same membrane strip can be used directly for automated Edman degradation. Comparable initial and repetitive yields were obtained for blotted peptides with and without matrix incubation.

Chromatography, High Pressure Liquid↗
PubMed1995

[Serratia marcescens nucleases. II. Primary structure analysis by peptide mapping combined with plasma-desorption mass spectrometry].

The primary structures of nucleases Sm1, Sm2, and Sm3 produced by Serratia marcescens were completely characterized using plasma desorption mass spectrometry (PDMS) of proteolytic peptides isolated by reverse-phase HPLC. The isoforms were separated by anion-exchange chromatography on DEAE cellulose and subjected to hydrolysis by the lysine-specific endoproteinase Lys-C. Comparative analysis of the peptides identified by PDMS showed that all three nucleases are N-terminal variants of the same protein: Sm2 represents a "mature" protein form, whereas Sm1 and Sm3 lack three and one N-terminal amino acid residues, respectively.

Amino Acid Sequence↗
PubMed1990

Peptide mapping.

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Autoradiography↗