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.

Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics

“…In cases where isoform- and PTM-directed antibodies are available along with knockout cells and/or mutant constructs, peak identity has been determined [23, 46, 47, 50]. Mass spectrometry (MS) peak identification protocols have also been developed and used for identification of previously unknown phosphorylation sites on their targets in the CNIA system [44, 45, 81]. For broader application, peak ID protocols will need to be developed for more complex PTM patterns.…”

“…Interested readers may refer to Libeler’s recent review [84] about MRM for more information. As mentioned in the previous session that MS protocols have been used as a strategy to detect the detail identity of PTM isoforms revealed by the charge-CNIA assays [44, 45, 81]. Integrating the different analysis methods will allow a more comprehensive study of proteins and their functioning mechanisms and will help translate the discovery research data into clinical practice.…”

Magneto-immunocapture with on-bead fluorescent labeling of amyloid-β peptides: towards a microfluidized-bed-based operation

“…Downscaling of this approach to microchip platforms has also been reported by our group with detectable levels of Aβ peptides close to 200 nM. 10,26,29,30 This technique belongs to the sample treatment strategies that are based on bio-functionalized magnetic nanoparticles. Recently Wiltfang's group presented a novel method based on capillary isoelectric focusing (CIEF) immunoassay which was capable of detecting total Aβ in CSF after desalting/ buffer exchange.…”

“…13 Esselmann et al described in their patent the use of quantitative ratio of Aβ 1-42, Aβ 2-40 and Aβ 2-42 for early diagnosis of AD. 11,[24][25][26] Like the two other techniques, the immunoassay based ones (MSD, xMAP, ELISA) can now detect different AD's biomarkers at the same time as they can employ simultaneously different antibodies specific for each biomarker. 11,[24][25][26] Like the two other techniques, the immunoassay based ones (MSD, xMAP, ELISA) can now detect different AD's biomarkers at the same time as they can employ simultaneously different antibodies specific for each biomarker.…”

“…Capillary zone electrophoresis (CZE), being the simplest mode of this technique, was applied successfully for separation and quantification of a mixture of different Aβ peptides down to 300-500 nM with UV detection 28 and 35 nM with laser-induced fluorescent detection (LIF), 10 respectively. 26 This method nevertheless is not applicable to C-truncated peptides possessing the same isoelectric point such as Aβ 1-38, Aβ 1-40 and Aβ 1-42. 9,29 These detection limits however were not sufficient for analyses of CSF samples where concentrations of Aβ peptides are only at sub nM ranges.…”