Micropurification of Two Human Cerebrospinal Fluid Proteins by High Performance Electrophoresis Chromatography

Abstract: Using C8 reversed-phase HPLC in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have fractionated proteins contained in human CSFs obtained from patients with schizophrenic disorders. When these proteins were electrophoretically blotted onto polyvinylidene difluoride membrane for direct N-terminal amino acid sequencing, several CSF proteins were identified; these included albumin, transferrin, apolipoprotein A-l, β₂-microglobulin, and prealbumin. We have also identified two structurally related human CSF proteins designated cerebrin 28 (M_r 28,000) and cerebrin 30 (M_r 30,000) that have an N-terminal amino acid sequence of NH₂-APPAQVSVQPNF and NH₂-APEAQVSVQPLFXQ, respectively. Comparison of these sequences with existing database at Protein Identification Resource (R 32.0), GenBank (R 72.0), SWISS-PROT (R 22.0), and EMBL (R 31.0) indicated that they are unique proteins. These proteins were subsequently purified by high performance electrophoresis Chromatography (HPEC) using an Applied Biosystems 230A HPEC system. A specific polyclonal antibody was prepared and an ELISA was established for cerebrin 30. It was noted that HPEC is a powerful tool to purify microgram quantities of proteins from human, rabbit, and rat CSFs. Using such a system, we have been able to micropurify as many as 10 proteins simultaneously in a single experiment because the elution of proteins occurred strictly according to their molecular weights. More importantly, we routinely obtained a recovery of >90%. The potential use of this technology for micropurification of proteins was discussed.