title: Analysis of amino-terminal variants of amyloid-β peptides by capillary isoelectric focusing immunoassay.
abstract: Here we present a novel assay for the separation and detection of amino-terminal amyloid-β (Aβ) peptide variants by capillary isoelectric focusing (CIEF) immunoassay. Specific amino-terminally truncated Aβ peptides appear to be generated by β-secretase (BACE1)-independent mechanisms and have previously been observed in cerebrospinal fluid (CSF) after BACE1 inhibitor treatment in an animal model. CIEF immunoassay sensitivity is sufficient to detect total Aβ in CSF without preconcentration. To analyze low-abundance amino-terminally truncated Aβ peptides from cell culture supernatants, we developed a CIEF-compatible immunoprecipitation protocol, allowing for selective elution of Aβ peptides with very low background. CIEF immunoassay and immunoprecipitation mass spectrometry analysis identified peptides starting at residue Arg(5) as the main amino-terminal Aβ variants produced in the presence of tripartite BACE1 inhibitor in our cell culture model. The CIEF immunoassay allows for robust relative quantification of Aβ peptide patterns in biological samples. To assess the future possibility of absolute quantification, we have prepared the Aβ peptides Aβ(x-10), Aβ(x-16), and Aβ(5-38(D23S)) by using solid phase peptide synthesis as internal standards for the CIEF immunoassay.
authors.affiliation: LVR-Klinikum Essen, Department of Psychiatry and Psychotherapy, Faculty of Medicine, University of Duisburg-Essen, D-45147 Essen, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Haußmann
authors.forename: Olaf
authors.initials: O
authors.lastname: Jahn
authors.forename: Philipp
authors.initials: P
authors.lastname: Linning
authors.forename: Christin
authors.initials: C
authors.lastname: Janßen
authors.forename: Thomas
authors.initials: T
authors.lastname: Liepold
authors.forename: Erik
authors.initials: E
authors.lastname: Portelius
authors.forename: Henrik
authors.initials: H
authors.lastname: Zetterberg
authors.forename: Chris
authors.initials: C
authors.lastname: Bauer
authors.forename: Johannes
authors.initials: J
authors.lastname: Schuchhardt
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Knölker
authors.forename: Hans
authors.initials: H
authors.lastname: Klafki
authors.forename: Jens
authors.initials: J
authors.lastname: Wiltfang
mesh_terms: D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000235:genetics; D002197:Capillary Action; D045744:Cell Line, Tumor; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007525:Isoelectric Focusing / Q000379:methods*; D008969:Molecular Sequence Data
chemical_list: D016229:Amyloid beta-Peptides