Analysis of Amino-Terminal Variants of Amyloid-beta Peptides by Capillary Isoelectric Focusing Immunoassay

作者：Haussmann Ute; Jahn Olaf; Linning Philipp; Janssen Christin; Liepold Thomas; Portelius Erik; Zetterberg Henrik; Bauer Chris; Schuchhardt Johannes; Knoelker Hans Joachim; Klafki Hans; Wiltfang Jens

来源：Analytical Chemistry, 2013, 85(17): 8142-8149.

DOI：10.1021/ac401055y

Here we present a novel assay for the separation and detection of amino-terminal amyloid-beta (A beta) peptide variants by capillary isoelectric focusing (CIEF) immunoassay. Specific amino-terminally truncated A beta peptides appear to be generated by beta-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 A beta in CSF without preconcentration: To analyze low-abundance amino-terminally truncated A beta peptides from cell culture supernatants, we developed a CIEF-compatible immunoprecipitation protocol, allowing for selective elution of A beta peptides with very low background. CIEF immunoassay and immunoprecipitation mass spectrometry analysis identified peptides starting at residue Arg(5) as the main amino-terminal A beta variants produced in the presence of tripartite BACE1 inhibitor in our cell culture model. The CIEF immunoassay allows for robust relative quantification of A beta peptide patterns in biological samples. To assess the future possibility of absolute quantification, we have prepared the A beta peptides A beta(x-10), A beta(x-16), and A beta(5-38(D23S)) by using solid phase peptide synthesis as internal standards for the CIEF immunoassay.

• 出版日期2013-9-3