Proteomics Analysis by 6-Plex TMT-LC/LC-MS/MS

The analysis was performed essentially based on the previously reported protocol (Bai et al., 2017; Niu et al., 2017). In brief, hippocampus was dissected from male B6J and D2 mice to create pooled samples consisting of three animals each. Pooled samples represented B6J mice aged approximately 60 days (two pooled samples) or 1 year (one pooled sample) and D2 mice aged 90 days (two pooled samples) or 1 year (one pooled sample). Tissue from pooled samples was lysed and digested into peptides. After desalting, the peptides were labeled with TMT reagents and equally mixed. The labeled samples were further fractionated by neutral pH reverse phase liquid chromatography (LC). A total of 10 fractions were collected and further analyzed by low pH reverse phase LC, and each fraction was analyzed by acidic pH reverse phase LC-MS/MS.

Collected data was searched against a database to identify peptides and filtered to achieve a 1% protein false discovery rate. TMT intensities were extracted, filtered, normalized, and summarized into peptide and protein quantification results. A total of 22,005 proteins representing 7,074 protein groups were identified and 7,014 protein groups were quantified. Statistical analysis was performed to determine cutoff for altered proteins and to evaluate associated false discovery rate. In this case the significance threshold was set at p < 0.005 with a minimum of 3 peptides detected. A total of 8 peptides and 34 spectral counts were detected for GABRA2 (full data set provided in Supplementary Table S1).