Figure 1

Experimental schematic for stable isotope labeling and isolation of mouse brain proteins. (A) Cohorts of mice were pulse-labeled with ¹³ C₆-leucine via a bolus intraperitoneal injection (200 mg/kg of body weight). After a pre-determined time following the ¹³ C₆-leucine administration, the mice were euthanized and the brains were removed. The brain tissue was then lysed using a 1% Triton X-100 lysis buffer, and the protein of interest was immunoprecipitated from the brain lysate (apoE is shown as an example). The precipitated proteins were then eluted from the antibody beads and subjected to trypsin digestion. The resulting peptide mixture was separated and analyzed via ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) (yellow = apoE, blue = sepharose bead, red = trypsin). (B) To observe the bioavailability of the ¹³ C₆-leucine, plasma samples were collected at sequential time points following the bolus injection and subjected to GC-MS analysis. The tracer-to-tracee ratio (TTR, shown as labeled/unlabeled leucine) was then measured by quantifying the relative amounts of ¹³ C₆-leucine and dividing by the amount of unlabeled leucine in each sample. Each time point in the graph represents the average value from 5–6 individual mice