Micropurification of two human cerebrospinal fluid proteins by high performance electrophoresis chromatography.
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-I, beta 2-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.
Leone
M G
MG
Population Council, New York, New York 10021.
Saso
L
L
Del Vecchio
A
A
Mo
M Y
MY
Silvestrini
B
B
Cheng
C Y
CY
D000595:Amino Acid Sequence; D000818:Animals; D002556:Cerebrospinal Fluid Proteins / Q000302:isolation & purification*; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000134:cerebrospinal fluid / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012559:Schizophrenia / Q000134:cerebrospinal fluid
D002556:Cerebrospinal Fluid Proteins; D009419:Nerve Tissue Proteins; C082261:cerebrin 28; C082262:cerebrin 30