Analysis of amino-terminal variants of amyloid-ß peptides by capillary isoelectric focusing immunoassay.

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