Tethered DNA hairpins facilitate electrochemical detection of DNA ligation

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

Glycoproteomic Analysis of Human Fibrinogen Reveals Novel Regions of O‑Glycosylation

Human fibrinogen is a 340 kDa, soluble plasma glycoprotein composed of paired sets of three subunits (α, β, γ). The protein plays a crucial role in protecting the vascular network against the loss of blood after tissue injury. The beta and gamma subunits each contain one N-glycosylation site, each of which is occupied by a biantennary N-glycan. So far O-linked oligosaccharides have rarely been described. Here, we make use of tryptic- and proteinase K-generated fibrinogen glycopeptides for the detailed analysis of the protein’s O-glycosylation by combining information obtained from both one- and two-dimensional nanoLC–ESI-ion trap (IT)–MS approaches. Glycopeptides were analyzed by ion trap-MS/MS which displayed fragmentations of glycosidic linkages and some peptide backbone cleavages. MS³ spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N-glycan attachment sites of human fibrinogen could be confirmed. Moreover, we describe seven novel O-glycosylation regions in human fibrinogen, all occupied by a monosialylated T-antigen. Our findings may help to improve the general understanding of human fibrinogen in the blood clotting process

Glycoproteomic Analysis of Human Fibrinogen Reveals Novel Regions of O‑Glycosylation

Human fibrinogen is a 340 kDa, soluble plasma glycoprotein composed of paired sets of three subunits (α, β, γ). The protein plays a crucial role in protecting the vascular network against the loss of blood after tissue injury. The beta and gamma subunits each contain one N-glycosylation site, each of which is occupied by a biantennary N-glycan. So far O-linked oligosaccharides have rarely been described. Here, we make use of tryptic- and proteinase K-generated fibrinogen glycopeptides for the detailed analysis of the protein’s O-glycosylation by combining information obtained from both one- and two-dimensional nanoLC–ESI-ion trap (IT)–MS approaches. Glycopeptides were analyzed by ion trap-MS/MS which displayed fragmentations of glycosidic linkages and some peptide backbone cleavages. MS³ spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N-glycan attachment sites of human fibrinogen could be confirmed. Moreover, we describe seven novel O-glycosylation regions in human fibrinogen, all occupied by a monosialylated T-antigen. Our findings may help to improve the general understanding of human fibrinogen in the blood clotting process

Glycoproteomic Analysis of Human Fibrinogen Reveals Novel Regions of O‑Glycosylation

Human fibrinogen is a 340 kDa, soluble plasma glycoprotein composed of paired sets of three subunits (α, β, γ). The protein plays a crucial role in protecting the vascular network against the loss of blood after tissue injury. The beta and gamma subunits each contain one N-glycosylation site, each of which is occupied by a biantennary N-glycan. So far O-linked oligosaccharides have rarely been described. Here, we make use of tryptic- and proteinase K-generated fibrinogen glycopeptides for the detailed analysis of the protein’s O-glycosylation by combining information obtained from both one- and two-dimensional nanoLC–ESI-ion trap (IT)–MS approaches. Glycopeptides were analyzed by ion trap-MS/MS which displayed fragmentations of glycosidic linkages and some peptide backbone cleavages. MS³ spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N-glycan attachment sites of human fibrinogen could be confirmed. Moreover, we describe seven novel O-glycosylation regions in human fibrinogen, all occupied by a monosialylated T-antigen. Our findings may help to improve the general understanding of human fibrinogen in the blood clotting process