The confirmation human brain proteomes were profiled from the dPFC of post-mortem brain samples from 198 participants of European descent recruited by the Banner Sun Health Research Institute (Banner). Participants in this study were recruited from the retirement communities in the greater Phoenix, Arizona, USA. All enrolled participants or their legal representatives signed an informed consent and the study was approved by the Institutional Review Board of Banner Sun Health Research Institute. Participants consented to annual standardized medical, neurological, and neuropsychological testing. Research diagnoses were made using approved research guidelines and a final clinicopathological diagnosis was made after review of all clinical, medical records, and neuropathological findings6 (link). Only subjects with a final diagnosis of normal cognition or AD were included in the proteomic analysis. Proteomic profiling was performed using the same approach as described above for the discovery proteomes with two differences: only MS2 scans were obtained and MS2 spectra were searched against the UniProtKB human brain proteome database downloaded in April 2015. Due to different databases, exact Uniprot IDs were used when comparing the discovery and confirmation results. In total, there were 11,518 proteins quantified. We applied the same quality control procedure as was done in the discovery proteomic dataset to the confirmation proteomic data. Likewise, we used regression to remove the effects of proteomic sequencing batch, age, sex, post-mortem interval, and final clinical diagnosis of cognitive status from the confirmatory proteomic profiles before estimating the protein weights.
Genotyping was performed using the Affymetrix Precision Medicine Array using DNA extracted from the brain with the Qiagen GenePure kit. We applied the same approach to quality control as described for the discovery dataset, including removing individuals based on data completeness or relatedness, removing sites with evidence of deviation from Hardy Weinberg equilibrium, missingness above 5%, minor allele frequency below 1%, or are not a SNP. Genotyping was imputed to the 1000 Genome Project Phase 326 (link) using the Michigan Imputation Server27 (link). SNPs with imputation R2> 0.3 were retained. Finally, only sites included in the linkage disequilibrium reference panel were used in our confirmation PWAS, as recommended by the FUSION pipeline. After quality control, there were 152 subjects with both proteomic and genetic data to include in our confirmation analyses.
Genotyping was performed using the Affymetrix Precision Medicine Array using DNA extracted from the brain with the Qiagen GenePure kit. We applied the same approach to quality control as described for the discovery dataset, including removing individuals based on data completeness or relatedness, removing sites with evidence of deviation from Hardy Weinberg equilibrium, missingness above 5%, minor allele frequency below 1%, or are not a SNP. Genotyping was imputed to the 1000 Genome Project Phase 326 (link) using the Michigan Imputation Server27 (link). SNPs with imputation R2> 0.3 were retained. Finally, only sites included in the linkage disequilibrium reference panel were used in our confirmation PWAS, as recommended by the FUSION pipeline. After quality control, there were 152 subjects with both proteomic and genetic data to include in our confirmation analyses.
