Identification of the apolipoprotein E4 isoform in cerebrospinal fluid with preparative two-dimensional electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Apolipoprotein E (apoE) was isolated from human cerebrospinal fluid (CSF) f rom control individuals and patients with Alzheimer's disease (AD). The pur ification was performed with preparative two-dimensional electrophoresis (2 -DE), involving liquid-phase isoelectric focusing (IEF) in the Rotofor cell in combination with sodium dodecyl sulfate polyacrylamide gel electrophore sis (SDS-PAGE) and electroelution in the Mini Whole Gel fluter. ApoE was ch aracterized by matrix-assisted laser desorption/ionization-time of flight-m ass spectrometry (MALDI-TOF-MS) analysis of tryptic digests. The known chan ge of Cys to Arg in position 112 of the apoE4 isoform was identified. This was detected in CSF from AD patients, reflecting the increased frequency of the apoE4 allele in this population. This peptide was not detected in CSF samples from healty control individuals. The use of this rapid electrophore tic separation in proteomic studies of CSF proteins provides single protein s, such as apoE, of high purity in yields sufficient for characterization b y MALDI-TOF-MS. Characterization of proteins and their modifications (amino acid substitutions, glycosylation or phosphorylation) in CSF will be a use ful tool in the investigation of the pathophysiology of brain disorders suc h as AD.