Case Western Reserve University Dublin, Ohio, United States
Eyad Hamza (Case Western Reserve University)| Sausan Azzam (Case Western Reserve University, Case Western Reserve University)| Kingman Strohl (Case Western Reserve University, Case Western Reserve University)
Objectives: Apolipoprotein A2 (ApoA2) gene expression is deterministic of apnea and have broad effects on breathing and fictive brainstem phrenic rhythm stability (Gillombardo et al, 2016 and 2017). ApoA2 is the second most abundant apolipoprotein in the circulation, however, there is a lack of fundamental information relevant to its presence and actions in the brain. Our previous data showed no ApoA2 protein in the KO mouse; and a lesser abundance of the protein in WT brain in comparison to either WT liver or plasma (P=0.0001). To gain a mechanistic understanding of the systems-level perturbations mediated by ApoA2 presence (ApoA2-wild type, WT) or absence (ApoA2 knockout, KO), we performed a quantitative proteomics and pathway analyses.
Methods: Brain tissue samples (WT, n= 8; KO, n=8) were lysed, FASPed, and trypsin digested (Azzam et al 2016 and 2017). Each sample was then analyzed by LC-MS/MS using LTQ Orbitrap Elite mass spectrometer (MS). Differential quantification of peptides was performed using Rosetta Elucidator quantitative software. Peptide and protein assignments were made using peptide and protein teller algorithm (FDR <2%). Analysis of variance (ANOVA) was performed to calculate the P-value Protein fold changes (FC) were determined by dividing the average protein intensity for each peptide in each group. Significant proteins (P-value ≤ 0.05 and FC with ≤-2 and ≥+2) were imported into Ingenuity Pathway Analysis (IPA) for network and pathway analyses. Protein expression of selected target proteins was validated using Selected Reaction Monitoring (SRM)-MS.
Results: A total of 6307 peptides were identified and quantified. Of those, 1150 peptides mapping to 298 unique proteins were differentially expressed across groups based on three cutoff criteria (P-value ≤0.05, two or more peptides per protein, and fold change of 2 or greater). IPA analysis identified mitochondrial function (oxidative phosphorylation, citric acid cycle and respiratory electron transport) as the top dysregulated canonical pathway. Furthermore, alteration of mitochondrial proteins including ATP-citrate synthase, V-type proton ATPase subunit C1, cytochrome c oxidase subunit 5A, and cytochrome b-c1 complex subunit 2 was confirmed by SRM analysis. This finding is consistent with our previous quantitative proteomic and pathway analyses of liver tissue that have indicated an effect of ApoA2 on mitochondrial electron transport chain.
Conclusions: Quantitative proteomics and pathway analyses identify perturbations in mitochondrial signaling as a consequence of ApoA2 loss with implication for energy homeostasis and cellular bioenergetics systems in the brain.