Study of the feasibility of using a pellicular anion-exchange column for separation of transferrin isoforms in human serum by HPLC with UV detection

Abstract

A method has been optimised for the separation of glycoforms of human serum transferrin, using a high-performance pellicular anion-exchange chromatographic column. The effect of the eluent pH and of the column temperature on the separation of transferrin glycoforms was studied using a standard solution of commercially available human serum transferrin. An HPLC system equipped with an ultraviolet detector was used for the analysis. No immunoassay was used after the anion-exchange chromatographic separation of the glycoforms, in contrast with most currently used methods. The method was applied to the separation and quantification of transferrin glycoforms in serum from a healthy, non-pregnant woman, after saturation of transferrin with iron and further precipitation of lipoproteins. The whole chromatographic run, including re-equilibration of the column, took 35 min.

Chromatographic and electrophoretic characterization of protein variants

“…An analytical method for the separation of glycoforms, such as human serum transferrin, differing in their amount of sialic acid residues by AIEX (Dionex ProPac PA1 column) was shown by De La Calle Guntiñas et al [53]. In addition to the commonly applied salt-gradients in IEC, pH-gradient elution is an indispensable method for the separation of variants due to charge heterogeneity.…”

Analysis of Nickel-Binding Peptides in a Human Hepidermoid Cancer Cell Line by Ni-NTA Affinity Chromatography and Mass Spectrometry

“…Most of these proteins are enzymes that require for their catalytic activity the presence of metal ions that could act as primary catalytic centre, or could form coordination complex in binding both the substrate and enzyme [2]. In addition, the interaction of proteins with metal may also be essential in the stabilization of protein structural domains.…”

Speciation of protein‐bound trace elements by gel electrophoresis and atomic spectrometry

“…Szpunar [1] has comprehensively reviewed the field of bioinorganic speciation analysis using hyphenated analytical systems. Articles on characterization of metal-binding proteins by high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are also available [2,3].…”