RESUMEN
During the course of studies on the analysis of O-glycans in biological samples, we found that significant amount of free glycans are present in normal human serum samples. The most abundant free glycan was disialo-biantennary glycan typically observed in transferrin which is one of the abundant glycoproteins found in sera. Minor glycans were also considered to be mainly due to transferrin, but some glycans were derived from mucin-type O-glycans, although the amount was quite minute. However, high mannose-type glycans could not be detected at all. Although there have been many reports on the presence of intracellular "free" N-glycans (mainly derived from high mannose-type glycans) generated either from lipid-linked oligosaccharides or from misfolded glycoproteins through endoplasmic-reticulum associated protein degradation pathway, there is little information on the presence of free glycans in extracellular matrix and biological fluids such as serum. This report is the first one which demonstrates the presence of free glycans due to glycoproteins in sera.
Asunto(s)
Glicoproteínas/química , Polisacáridos/análisis , Polisacáridos/sangre , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión/métodos , Glicoproteínas/sangre , Humanos , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de Electrospray/métodos , Transferrina/químicaRESUMEN
The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.
Asunto(s)
Productos Biológicos/química , Monosacáridos/análisis , Amino Azúcares/análisis , Amino Azúcares/normas , Productos Biológicos/normas , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/normas , Cromatografía por Intercambio Iónico/métodos , Cromatografía por Intercambio Iónico/normas , Eritropoyetina/química , Excipientes , Glicosilación , Monosacáridos/normas , Proteínas Recombinantes , Estándares de Referencia , Reproducibilidad de los Resultados , Ácidos Siálicos/análisis , Ácidos Siálicos/normas , Activador de Tejido Plasminógeno/químicaRESUMEN
An incomplete elongation of O-glycans in mucins has been found in epithelial cancers, leading to the expression of shorter carbohydrate structures such as Tn antigen (GalNAc-O-Ser/Thr), which has been reported to be one of the most specific human cancer-associated structures. However, there have been no appropriate physicochemical methods for the determination of Tn antigen in biological samples. In the present paper, we developed a capillary electrophoresis method for the determination of Tn antigen, and applied the method to the analysis of the expressed Tn antigen on some leukemia and epithelial cancer cells.
