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) from control individuals and patients with Alzheimer's disease (AD). The purification 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 electrophoresis (SDS-PAGE) and electroelution in the Mini Whole Gel Eluter. ApoE was characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of tryptic digests. The known change 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 electrophoretic separation in proteomic studies of CSF proteins provides single proteins, such as apoE, of high purity in yields sufficient for characterization by MALDI-TOF-MS. Characterization of proteins and their modifications (amino acid substitutions, glycosylation or phosphorylation) in CSF will be a useful tool in the investigation of the pathophysiology of brain disorders such as AD.
Hesse
C
C
Department of Clinical Neuroscience, Göteborg University, Sahlgrenska University Hospital/Mölndal, Sweden. camilla.hesse@neuro.gu.se
Nilsson
C L
CL
Blennow
K
K
Davidsson
P
P
D000328:Adult; D053327:Apolipoprotein E4; D001057:Apolipoproteins E / Q000134:cerebrospinal fluid*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D008875:Middle Aged; D020033:Protein Isoforms / Q000134:cerebrospinal fluid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D053327:Apolipoprotein E4; D001057:Apolipoproteins E; D020033:Protein Isoforms