Fig. 6. Rates of Aβ synthesis do not differ according to human apoE isoform in PDAPP/TRE mice.

(A) Aβ detection in hippocampal lysates from young PDAPP/TRE mice by TSQ Vantage triple quadrupole mass spectrometry. Left, representative total ion count multiple reaction monitoring (MRM) peak of the unlabeled Aβ tryptic peptide, LVFFAEDVGSNK, (m/z = 663.340). Right, MRM peak for [¹³C₆]-leucine labeled Aβ (m/z = 666.350). (B) Standard curve generated with known quantity of [¹³C₆]-leucine labeled and unlabeled Aβ. Aβ secreted from H4-APP695ΔNL neuroglioma cells incubated with labeled/unlabeled leucine was immunoprecipitated with HJ5.2 antibody (anti-Aβ_13-28), followed by trypsin digestion. Aβ_17-28 fragments were analyzed on a TSQ Vantage mass spectrometer. The expected percentage of labeled Aβ versus measured percentage was fit by linear regression. Variance is reported with 95% confidence interval. (C) Relative FSRs of Aβ from hippocampi of PDAPP/TRE mice intraperitoneally injected with [¹³C₆]-leucine (200mg/kg) (n=5 to 6 mice per group; 4 to 5 months old). Relative FSRs of Aβ were calculated from the ratio of [¹³C₆]-leucine labeled to unlabeled Aβ. [¹³C₆]/[¹²C₆]-Aβ ratio was normalized to the ratio of labeled to unlabeled free leucine in plasma (determined by GC-MS). Mass spectrometry data were normalized with the media standard curve in (B). One-way ANCOVA (analysis of covariance) revealed no significant differences among groups. Values represent means ± SEM.