Use of hydrophobic interaction methods in the isolation of proteins from endocrine and paraendocrine tissues and cells by high-performance liquid chromatography

Abstract

We have recently described the separation of a large number of polypeptide hormones, related peptides and some protein standards by hydrophobic interaction high-performance liquid chromatography (HPLC). This paper reports the practical application of these methods to the reproducible isolation and separation of components of a mixture of immunoreactive calcitonin-like proteins (less than 25 kD) synthesised and secreted by human tumour cells in vitro. Using hydrophobic interaction HPLC on ODS-silica for both preliminary bulk fractionation and subsequent analytical separation greater than 80% recoveries of small (ng) quantities of immunoreactive proteins were obtained from samples containing less than 100 mg total protein, and characteristic profiles of synthesised and secreted materials were established. Using a partially purified hypothalamic extract, containing a number of small proteins (12--25 kD), we have also examined the effects of varying chromatographic conditions in an attempt to modify the separations obtained with ODS-silica using an acid-saline-acetonitrile gradient elution system at ambient temperature, and achieve further resolution of its components. No useful selective effects were observed when temperature, organic modifier, gradient profile or hydrophobic stationary phase were altered. These techniques may not therefore be inherently capable of completely resolving all components of natural protein mixtures. They do, however, offer an adjunct to and in certain cases a substitute for conventional methods of protein separation.