RESUMEN
Core-fucosylated glycans, derivatized with 2-aminoacridone, consistently migrate slower than the corresponding oligosaccharides which lack this fucose residue, using the micellar electrophoretic capillary chromatography conditions outlined in this study. alpha-Fucosidase digestion of glycans followed by CE analysis has allowed facile differentiation of these two classes of oligosaccharides and this methodology has been applied to obtain preliminary information on the carbohydrate content from two glycoproteins, a monoclonal IgG antibody and the soluble complement receptor type 1 (sCR1).
Asunto(s)
Electroforesis Capilar/métodos , Fucosa/análisis , Polisacáridos/análisis , Aminoacridinas , Conformación de Carbohidratos , Secuencia de Carbohidratos , Colorantes Fluorescentes , Inmunoglobulina G/química , Datos de Secuencia Molecular , Receptores de Complemento/química , Proteínas Recombinantes/química , alfa-L-Fucosidasa/metabolismoRESUMEN
By digestion of the highly basic polypeptide aprotinin or bovine pancreatic trypsin inhibitor (BPTI) with endoproteinase Lys-C after unfolding, reduction and pyridylethylation, five fragments are obtained. These fragments are separated by free solution capillary electrophoresis using a phosphate buffer at neutral pH. The effect of the ion-pairing buffer additive phytic acid on the separation was investigated. It is shown that phytic acid through ion-pair formation influences the mobility of only those peptide fragments having a net positive charge at the pH of the separation buffer. The affinity of phytic acid to the peptides correlates with their isoelectric point and the charge to mass ratios. Hence, by changing the concentration of phytic acid, it is possible to manipulate the migration order and the separation of the peptides.
Asunto(s)
Electroforesis Capilar/métodos , Mapeo Peptídico/métodos , Ácido Fítico/química , Secuencia de Aminoácidos , Aprotinina/química , Iones , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Mapeo Peptídico/normasRESUMEN
The addition of the sodium salt of phytic acid to the separation buffer (pH's 6.0-9.5) has allowed the analysis of a number of basic proteins (pI's > 9) by capillary electrophoresis. The method of analysis is simple and leads to considerable improvement in peak shape. Some very basic proteins, totally adsorbed onto the capillary fused silica surfaces in the presence of buffer only, can be analysed as sharp signals when this polyanionic species is included in the running electrolyte. These improvements in analysis are thought to arise as a result of the suppression of coulombic interactions between these positively charged proteins (ion-paired to phytic acid) and the negatively charged silanol groups on the inner wall of the capillary.
Asunto(s)
Electroforesis Capilar/métodos , Proteínas/aislamiento & purificación , Adsorción , Tampones (Química) , Electroquímica , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Ácido Fítico/farmacologíaRESUMEN
Capillary electrophoresis can be applied to the rapid characterization of tryptic digests of proteins. The addition of phytic acid to the separation buffer was found to improve resolution considerably when the technique was applied to differentiate between tryptic digests derived from variant haemoglobins. Moreover, analysis time was of the order of 15 min, which is considerably shorter than that obtained using gradient reversed-phase high-performance liquid chromatography or two-dimensional paper chromatography-electrophoresis.
Asunto(s)
Electroforesis/métodos , Hemoglobinas/química , Fragmentos de Péptidos/análisis , Ácido Fítico , Animales , Cromatografía Líquida de Alta Presión/métodos , Cromatografía en Papel/métodos , Variación Genética , Hemoglobinas/genética , Humanos , Mapeo PeptídicoRESUMEN
Studies are reported on the effect of the sodium salt of phytic acid on the resolution of peptides and proteins. Improved separation in the case of peptides is shown to be due to ion-ion pairing interactions between the positively charged peptides and the phytic acid polyanionic species. The improved peak shapes related to the proteins can be interpreted in terms of the sample preconcentration due to injection of analytes from a water medium to one of high ionic strength.
Asunto(s)
Bradiquinina/aislamiento & purificación , Péptidos/aislamiento & purificación , Ácido Fítico , Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Aminoácidos/análisis , Bradiquinina/análogos & derivados , Bradiquinina/química , Acción Capilar , Electroforesis/métodos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Péptidos/química , Fenilalanina/análisis , Proteínas/química , Triptófano/análisisRESUMEN
A micellar electrokinetic capillary chromatographic method has been developed for the qualitative assay of amoxycillin and its degradation products and clavulanic acid. Together with amoxycillin the latter acid is an important constituent in the antibiotic Augmentin. The analytical procedure is fast and analytes can be identified both from their migration times and from changes in migration time observed either at different pH values or in electropherograms run in H2O and D2O based buffers of the same acidity.
Asunto(s)
Amoxicilina/análisis , Cromatografía/métodos , Electroquímica/métodos , Electroforesis/métodos , MicelasRESUMEN
The capillary electrophoresis (CE) of peptide fragments from the tryptic digest of salmon calcitonin and elcatonin has been carried out in H2O- and D2O-based buffer solutions. Analysis in heavy water was found to be superior to that in H2O especially at a pH or pD of 7.93. From a single CE run on elcatonin digest we were also able to harvest three pure cleavage peptides in sufficient quantity to determine each amino acid residue by protein sequencing. The order of elution from CE agreed with that predicted on the basis of net charge calculated for each peptide.
Asunto(s)
Calcitonina/química , Electroforesis/métodos , Péptidos/análisis , Secuencia de Aminoácidos , Animales , Calcitonina/análogos & derivados , Deuterio/metabolismo , Óxido de Deuterio , Datos de Secuencia Molecular , Salmón , Tripsina/metabolismo , Agua/metabolismoRESUMEN
In this study we have explored the behaviour of peptides after capillary electrophoresis (CE) followed by elution under pressure. The use of D2O- rather than H2O-based buffer solutions appears to restrict the diffusion of peptides after CE, resulting in little loss of resolution when peptides are eluted by dynamic flow. In this paper we present results showing that a simple two-step process, involving CE at a low voltage, switching off the power supply, and connecting the fused capillary at the anode end to a syringe pump for dynamic flow, can retain separation characteristics and can be used for the isolation of picomole quantities of peptides for sequence determination.