RESUMO
Three different analytical techniques (planar SDS-PAGE, CGE-on-a-chip and MALDI-TOF-MS) applied for determination of the molecular weight of intact and partly and completely de-N-glycosylated human serum glycoproteins (antithrombin III and coagulation factor IX) have been compared. N-Glycans were removed from the protein backbone of both complex glycoproteins using PNGase F, which cleaves all types of asparagine-attached N-glycan provided the oligosaccharide has at least the length of a chitobiose core unit. Two of the applied techniques were based on gel electrophoretic separation in the liquid phase while the third technique was the gas-phase technique mass spectrometry. It was demonstrated that the enzymatic de-N-glycosylation generally worked well (completely or partially) with both glycoproteins (one containing only N-glycans and the second N- and O-glycans). All three methods were suitable for monitoring the de-N-glycosylation progress. While the molecular weights determined with MALDI-TOF-MS were most accurate, both gel electrophoretic methods provided molecular weights that were too high because of the attached glycan structures.
Assuntos
Anticoagulantes/análise , Antitrombina III/análise , Eletroforese Capilar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Fator IX/análise , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Anticoagulantes/metabolismo , Antitrombina III/metabolismo , Asparagina/química , Asparagina/metabolismo , Sequência de Carboidratos , Dissacarídeos/química , Dissacarídeos/metabolismo , Fator IX/metabolismo , Glicosilação , Humanos , Dados de Sequência Molecular , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Fatores de TempoRESUMO
Rapid screening for and selection of potential metabolites of urapidil in dog urine by high-performance liquid chromatographic analysis is achieved by use of a diode array UV detector, which allows multi-wavelength detection and collects several complete UV spectra from each peak in the course of a single chromatographic analysis. Because of the similarity of metabolite spectra to the spectrum of the parent compound, few interesting peaks are picked out, rather rapidly, by means of a selection criterion based on absorption ratios. Inspection of the complete UV spectra of these pre-selected peaks allows further restriction to "candidate" metabolite peaks. Comparison of up-slope, apex and down-slope spectra is a convenient way of testing for peak purity.