Application of quantitative LC–MS surrogate peptide methodology in the analysis of the amyloid beta peptide (Aβ) biosynthetic intermediate protein APP–βCTF

An area of current research in Alzheimer's disease (AD) is the biosynthetic pathway of amyloid beta peptides (Aβ) via consecutive proteolytic cleavages of the amyloid beta precursor protein (APP) by BACE and γ-secretase enzymes. APP is first cleaved by BACE to form a C-terminal fragment APP–βCTF, or also called C99, which then undergoes further cleavage by γ-secretase to form Aβ. Inhibitors of γ-secretase have been observed to yield a so-called ‘Aβ rise’ phenomenon whereby low inhibitor concentrations result in an increase in Aβ levels while high inhibitor concentrations result in lower Aβ levels. A previous report from our labs indicated that this phenomenon was related to ratios of APP–βCTF substrate relative to γ-secretase enzyme. A quantitative Western blot analysis was used with a recombinant C100 protein as calibration standards to assess the relationship of APP–βCTF, γ-secretase enzyme and various inhibitors resulting in the ‘Aβ rise’. An on-line liquid chromatography mass spectrometry (LC–MS) method employing the ‘surrogate peptide’ methodology was developed to accurately quantify the recombinant C100 used in the Western blot analyses. The surrogate peptide approach utilizes tryptic digestion of the protein to stoichiometrically yield a unique peptide fragment, in this case C100Aβ17–28 (LVFFAEDVGSNK) that can be readily detected by LC–MS. The absolute quantitative assessment of C100 was accomplished using synthetic Aβ17–28 to generate calibration curves over a 0.001–1 μM range and 15N isotopically labeled Aβ1–40 as the internal standard for enzymatic digestion and its proteolytic peptide [15N]-Aβ17–28 for the analysis.