Stroke is a leading cause of mortality and disability, and ischemic stroke accounts for more than 80% of the disease occurrence. Timely reperfusion is essential in the treatment of ischemic stroke, but it is known to cause ischemia-reperfusion (I/R) injury and the relevant studies have mostly focused on the acute phase. Here we reported on a global proteomic analysis to investigate the development of cerebral I/R injury in the subacute and long-term phases. A rat model was used, with 2 h-middle cerebral artery occlusion (MCAO) followed with 1, 7, and 14 days of reperfusion. The proteins of cerebral cortex were analyzed by SDS-PAGE, whole-gel slicing, and quantitative LC-MS/MS. Totally 5621 proteins were identified, among which 568, 755, and 492 proteins were detected to have significant dys-regulation in the model groups with 1, 7, and 14 days of reperfusion, respectively, when compared with the corresponding sham groups (n = 4, fold change ≥1.5 or ≤0.67 and p ≤ 0.05). Bioinformatic analysis on the functions and reperfusion time-dependent dys-regulation profiles of the proteins exhibited changes of structures and biological processes in cytoskeleton, synaptic plasticity, energy metabolism, inflammation, and lysosome from subacute to long-term phases of cerebral I/R injury. Disruption of cytoskeleton and synaptic structures, impairment of energy metabolism processes, and acute inflammation responses were the most significant features in the subacute phase. With the elongation of reperfusion time to the long-term phase, a tendency of recovery was detected on cytoskeleton, while inflammation pathways different from the subacute phase were activated. Also, lysosomal structures and functions might be restored. This is the first work reporting the proteome changes that occurred at different time points from the subacute to long-term phases of cerebral I/R injury and we expect it would provide useful information to improve the understanding of the mechanisms involved in the development of cerebral I/R injury and suggest candidates for treatment.

MS identification has long been used for PAGE-separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole-gel slicing and quantitative LC-MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty-five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in-gel digestion and quantitative LC-MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1-28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as "native MS-electropherograms". Comparison of the electropherograms revealed significant plasma-versus-serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.

Hyperlipidemia is a chronic disease characterized by elevated blood cholesterol and triglycerides and there is accumulated evidence that the disease might affect brain functions. Here we report on a proteomic analysis of the brain proteins in hyperlipidemic mice. Hyperlipidemia was successfully induced in mice by a 20 week high-fat diet (HFD) feeding (model group). A control group with a normal diet and a treatment group with HFD-fed mice treated with a lipid-lowering drug simvastatin (SIM) were established accordingly. The proteins were extracted from the left and right cerebrum hemispheres of the mice in the three groups and subjected to shotgun proteomic analysis. A total of 4,422 proteins were detected in at least half of the samples, among which 324 proteins showed significant difference (fold change >1.5 or <0.67, p < 0.05) in at least one of the four types of comparisons (left cerebrum hemispheres of the model group versus the control group, right cerebrums of model versus control, left cerebrums of SIM versus model, right cerebrums of SIM versus model). Biological process analysis revealed many of these proteins were enriched in the processes correlated with lipid metabolism, neurological disorders, synaptic events and nervous system development. For the first time, it has been reported that some of the proteins have been altered in the brain under the conditions of HFD feeding, obesity or hyperlipidemia. Further, 22 brain processes-related proteins showed different expression in the two cerebrum hemispheres, suggesting changes of the brain proteins caused by hyperlipidemia might also be asymmetric. We hope this work will provide useful information to understand the effects of HFD and hyperlipidemia on brain proteins.

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