The Use of High-Performance Electrophoresis Chromatography for the Micropurification of Cerebrospinal Fluid Proteins in the Rat

Abstract

Using reversed-phase high-performance liquid chromatography (HPLC) on a Vydac C8 column in conjunction with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and silver staining, we have identified more than 30 proteins in cerebrospinal fluid collected from adult rats by cannulation of cisterna magna. When these partially purified cerebrospinal fluid proteins were further fractionated by high-performance electrophoresis chromatography (HPEC) on an Applied Biosystems 230A HPEC system using a 10% T SDS-polyacrylamide gel with a phosphate base running buffer system under nonreducing conditions, we have purified more than 10 proteins to apparent homogeneity from a pool of 10 ml of rat cerebrospinal fluid as verified by silver staining and direct N-terminal amino acid sequencing. Two additional series of experiments using rat cerebrospinal fluid over a 12-month period yielded virtually identical results. A major advantage of HPEC over conventional HPLC is that the recovery of protein is almost quantitative and is in the range of 90-95% using as little as 1 μg of protein. The purified proteins from HPEC are ready for direct protein sequencing following a buffer exchange to remove residual Tris and phosphate without additional manipulation. The potential use of HPEC for micropurification of proteins was discussed.