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

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• 作者：Ute Hau脽mann ; Olaf Jahn ; Philipp Linning ; Christin Jan脽en ; Thomas Liepold ; Erik Portelius ; Henrik Zetterberg ; Chris Bauer ; Johannes Schuchhardt ; Hans-Joachim Kn枚lker ; Hans Klafki ; Jens Wiltfang
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：September 3, 2013
• 年：2013
• 卷：85
• 期：17
• 页码：8142-8149
• 全文大小：388K
• 年卷期：v.85,no.17(September 3, 2013)
• ISSN：1520-6882

文摘

Here we present a novel assay for the separation and detection of amino-terminal amyloid-尾 (A尾) peptide variants by capillary isoelectric focusing (CIEF) immunoassay. Specific amino-terminally truncated A尾 peptides appear to be generated by 尾-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尾 in CSF without preconcentration. To analyze low-abundance amino-terminally truncated A尾 peptides from cell culture supernatants, we developed a CIEF-compatible immunoprecipitation protocol, allowing for selective elution of A尾 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尾 variants produced in the presence of tripartite BACE1 inhibitor in our cell culture model. The CIEF immunoassay allows for robust relative quantification of A尾 peptide patterns in biological samples. To assess the future possibility of absolute quantification, we have prepared the A尾 peptides A尾_x-10, A尾_x-16, and A尾_5-38(D23S) by using solid phase peptide synthesis as internal standards for the CIEF immunoassay.