Quantitative Proteomic Analysis of G72Tg Mice

The hippocampal cytoplasmic fraction from G72Tg, WT and ¹⁵N-labeled CD1 mice was collected as previously described [9 (link)]. ¹⁵N-labeled CD1 mouse hippocampal tissue was used as internal standard so as to compare the unlabeled G72Tg and WT mice. The experimental design for the quantitative proteomics analysis is as previously described (Figure 1 in [9 (link)]). The Bradford assay (BioRad Laboratories, Hercules, CA, USA) was used to quantitate total protein amount. For each G72Tg/WT pair, the cytoplasmic fractions of G72Tg and WT hippocampi were mixed 1:1 (w/w) with the cytoplasmic fraction of the same ¹⁵N-labeled CD1 mouse reference based on protein amount. Proteomics sample preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in gel digestion and peptide extraction was performed as previously described [9 (link),10 (link)]. Peptide extracts were lyophilized, re-dissolved in 0.1% formic acid, filtered and analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) by a nanoflow HPLC-2D system (Eksigent, Dublin, CA, USA) which was coupled online to an LTQ Orbitrap XL™ Hybrid FT Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany), as previously described [9 (link)]. Detailed MS parameters were as previously described [12 (link)].