Methods in molecular biology (Clifton, N.J.), 2013
Liquid chromatography-tandem stage mass spectrometry of glycopeptides is a powerful tool for the ... more Liquid chromatography-tandem stage mass spectrometry of glycopeptides is a powerful tool for the site-specific glycosylation analysis of glycoproteins. Using fetuin as a model substance, we describe a protocol for glycopeptide dissection using nonspecific proteolysis by proteinase K. Proteolysis is achieved using dissolved or immobilized enzyme. For glycopeptide separation three different nanoHPLC separation principles are compared, namely hydrophilic interaction liquid chromatography (HILIC), C18-reverse phase (RP), and graphitized carbon HPLC. Chromatographically resolved glycopeptides are analyzed by nano-electrospray ionization multistage mass spectrometry for identification of the glycan as well as the peptide moiety. Using this approach, site-specific information on protein glycosylation is obtained.
Antimicrobial agents and chemotherapy, Jan 16, 2015
The worldwide-spread Escherichia coli clone ST131-O25b:H4 is responsible for a significant propor... more The worldwide-spread Escherichia coli clone ST131-O25b:H4 is responsible for a significant proportion of multi-drug resistant extraintestinal infections. We generated humanized monoclonal antibodies (mAbs) that target the lipopolysaccharide O25b antigen conserved within this lineage. These mAbs bound to the surface of live bacterial cells irrespective of the capsular type expressed. In a serum bactericidal assay in vitro, mAbs induced over 95% bacterial killing in the presence of human serum as complement source. Protective efficacy at low antibody doses was observed in a murine model of bacteremia. The mode of action in vivo was investigated by using aglycosylated derivatives of the protective mAbs. The significant binding to live E. coli cells as well as the in vitro and in vivo efficacy was corroborated in assays using bacteria grown in human serum to mimic relevant clinical conditions. Given the dry pipeline of novel antibiotics against multi-drug resistant Gram-negative pathoge...
Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of ... more Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component...
A novel electrochemical assay for DNA ligase activity is described. The assay exploits the proper... more A novel electrochemical assay for DNA ligase activity is described. The assay exploits the properties of DNA hairpins tethered at one terminus to a gold electrode and labelled at the other with a ferrocene group for rapid characterisation of DNA status by cyclic voltammetry. Successful ligation of 'nicked' DNA hairpins is indicated by retention of the ferrocene couple when exposure to DNA ligase is followed by conditions that denature the hairpin. The results demonstrate the simplicity of integrating electrochemical detection with hairpin based biosensors and illustrate a new approach to the assay of DNA ligases, of which the NAD(+)-dependent enzymes represent a potential broad spectrum antibacterial drug target.
Analysis of protein glycosylation is essential in order to correlate certain disease types with o... more Analysis of protein glycosylation is essential in order to correlate certain disease types with oligosaccharide structures on proteins. Here, a method for the MS characterization of site-specific protein glycosylation is presented. Using asialofetuin and fetuin as model substances, a protocol for glycopeptide dissection was developed based on unspecific proteolysis by Proteinase K. The resulting glycopeptides were then resolved by nanoscale hydrophilic interaction liquid chromatography-electrospray multistage MS. The early elution range of O-glycopeptides was clearly separated from the late elution range of N-glycopeptides. Glycopeptides were analyzed by ion trap-MS/MS, which revealed fragmentations of glycosidic linkages and some peptide backbone cleavages; MS(3) spectra predominantly exhibited cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N- and O-glycan attachment sites of fetuin could be confirmed; moreover using our method, the occupation of a new, additional O-glycosylation site serine 296 was found. In conclusion, this approach appears to be a valuable technique for in-depth analysis of the site-specific N-glycosylation and O-glycosylation of individual glycoproteins.
In this paper, the use of tyrosinase (Ty) from Streptomyces antibioticus, labeled with a fluoresc... more In this paper, the use of tyrosinase (Ty) from Streptomyces antibioticus, labeled with a fluorescent tag, in combination with soluble quinoprotein (PQQ-containing) glucose dehydrogenase (s-GDH) to measure trace amounts of phenols is explored. Proof of concept is provided by a series of experiments, which show a clear quantitative dependence of the response on the phenol concentration. One of the advantages of the detection system is that apart from a standard fluorimeter no further instrumentation is required.
Methods in molecular biology (Clifton, N.J.), 2013
Liquid chromatography-tandem stage mass spectrometry of glycopeptides is a powerful tool for the ... more Liquid chromatography-tandem stage mass spectrometry of glycopeptides is a powerful tool for the site-specific glycosylation analysis of glycoproteins. Using fetuin as a model substance, we describe a protocol for glycopeptide dissection using nonspecific proteolysis by proteinase K. Proteolysis is achieved using dissolved or immobilized enzyme. For glycopeptide separation three different nanoHPLC separation principles are compared, namely hydrophilic interaction liquid chromatography (HILIC), C18-reverse phase (RP), and graphitized carbon HPLC. Chromatographically resolved glycopeptides are analyzed by nano-electrospray ionization multistage mass spectrometry for identification of the glycan as well as the peptide moiety. Using this approach, site-specific information on protein glycosylation is obtained.
Antimicrobial agents and chemotherapy, Jan 16, 2015
The worldwide-spread Escherichia coli clone ST131-O25b:H4 is responsible for a significant propor... more The worldwide-spread Escherichia coli clone ST131-O25b:H4 is responsible for a significant proportion of multi-drug resistant extraintestinal infections. We generated humanized monoclonal antibodies (mAbs) that target the lipopolysaccharide O25b antigen conserved within this lineage. These mAbs bound to the surface of live bacterial cells irrespective of the capsular type expressed. In a serum bactericidal assay in vitro, mAbs induced over 95% bacterial killing in the presence of human serum as complement source. Protective efficacy at low antibody doses was observed in a murine model of bacteremia. The mode of action in vivo was investigated by using aglycosylated derivatives of the protective mAbs. The significant binding to live E. coli cells as well as the in vitro and in vivo efficacy was corroborated in assays using bacteria grown in human serum to mimic relevant clinical conditions. Given the dry pipeline of novel antibiotics against multi-drug resistant Gram-negative pathoge...
Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of ... more Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component...
A novel electrochemical assay for DNA ligase activity is described. The assay exploits the proper... more A novel electrochemical assay for DNA ligase activity is described. The assay exploits the properties of DNA hairpins tethered at one terminus to a gold electrode and labelled at the other with a ferrocene group for rapid characterisation of DNA status by cyclic voltammetry. Successful ligation of 'nicked' DNA hairpins is indicated by retention of the ferrocene couple when exposure to DNA ligase is followed by conditions that denature the hairpin. The results demonstrate the simplicity of integrating electrochemical detection with hairpin based biosensors and illustrate a new approach to the assay of DNA ligases, of which the NAD(+)-dependent enzymes represent a potential broad spectrum antibacterial drug target.
Analysis of protein glycosylation is essential in order to correlate certain disease types with o... more Analysis of protein glycosylation is essential in order to correlate certain disease types with oligosaccharide structures on proteins. Here, a method for the MS characterization of site-specific protein glycosylation is presented. Using asialofetuin and fetuin as model substances, a protocol for glycopeptide dissection was developed based on unspecific proteolysis by Proteinase K. The resulting glycopeptides were then resolved by nanoscale hydrophilic interaction liquid chromatography-electrospray multistage MS. The early elution range of O-glycopeptides was clearly separated from the late elution range of N-glycopeptides. Glycopeptides were analyzed by ion trap-MS/MS, which revealed fragmentations of glycosidic linkages and some peptide backbone cleavages; MS(3) spectra predominantly exhibited cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N- and O-glycan attachment sites of fetuin could be confirmed; moreover using our method, the occupation of a new, additional O-glycosylation site serine 296 was found. In conclusion, this approach appears to be a valuable technique for in-depth analysis of the site-specific N-glycosylation and O-glycosylation of individual glycoproteins.
In this paper, the use of tyrosinase (Ty) from Streptomyces antibioticus, labeled with a fluoresc... more In this paper, the use of tyrosinase (Ty) from Streptomyces antibioticus, labeled with a fluorescent tag, in combination with soluble quinoprotein (PQQ-containing) glucose dehydrogenase (s-GDH) to measure trace amounts of phenols is explored. Proof of concept is provided by a series of experiments, which show a clear quantitative dependence of the response on the phenol concentration. One of the advantages of the detection system is that apart from a standard fluorimeter no further instrumentation is required.
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Papers by Gerhild Zauner