Figure 2

Tandem mass spectrometry (MS/MS) analysis and quantitation of stable isotope labeled apoE. (A) Tryptic peptides from immunoprecipitated apoE were separated by liquid chromatography and detected using a Xevo TQ-S triple quadrupole mass spectrometer. To facilitate the accurate and specific quantitation of labeled apoE, MRM transitions and conditions were optimized for the parent ion LQAEIFQAR. MS/MS spectrum for the product MRM transitions is shown. A similar analysis was performed for the Aβ specific peptide. (B) Representative relative ion count peaks from multiple reaction monitoring (MRM) analysis of the labeled and unlabeled apoE parent peptide LQAEIFQAR are shown [mass charge ratio (m/z) = 541.29 for labeled peptide and 538.29 for unlabeled peptide]. The area under the curve of the MRM ion counts were used for quantitation of the labeled and unlabeled peptide (C) Standard curve of labeled apoE. To generate a standard curve for the MS quantitation, primary mouse astrocytes were incubated in culture media with different percentages of labeled leucine. The media of the astrocytes was then collected and the secreted apoE was immunoprecipitated, digested with trypsin, and analyzed using LC-MS. The measured labeled/unlabeled ratios along with the predicted labeled/unlabeled values are shown with a linear regression line (n = 3, dotted lines represent 95% confidence bands). (D) Representative diagram of time course of labeled proteins used for kinetic analyses. The labeled/unlabeled ratio was calculated for individual protein samples by dividing the area under the curve of the labeled ion count (IC) by the area under the curve of the unlabeled IC. The labeled/unlabeled ratios were averaged for all of the mouse brain samples collected at each time point. The averaged labeled/unlabeled ratios were then plotted versus time to obtain the kinetic curve for each mouse genotype.