Analysis of a membrane-enriched proteome from postmortem human brain tissue in Alzheimer's disease

Abstract

Purpose: The present study is a discovery mode proteomics analysis of the membrane-enriched fraction of postmortem brain tissue from Alzheimer's disease (AD) and control cases. This study aims to validate a method to identify new proteins that could be involved in the pathogenesis of AD and potentially serve as disease biomarkers.

Experimental design: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze the membrane-enriched fraction of human postmortem brain tissue from five AD and five control cases of similar age. Biochemical validation of specific targets was performed by immunoblotting.

Results: One thousand seven hundred and nine proteins were identified from the membrane-enriched fraction of frontal cortex. Label-free quantification by spectral counting and G-test analysis identified 13 proteins that were significantly changed in disease. In addition to Tau (MAPT), two additional proteins found to be enriched in AD, ubiquitin carboxy-terminal hydrolase 1 (UCHL1), and syntaxin-binding protein 1 (Munc-18), were validated through immunoblotting. DISCUSSION AND CLINICAL RELEVANCE: Proteomic analysis of the membrane-enriched fraction of postmortem brain tissue identifies proteins biochemically altered in AD. Further analysis of this subproteome may help elucidate mechanisms behind AD pathogenesis and provide new sources of biomarkers.

Figure 1. Membrane enrichment strategy

(A) Flowchart of experimental design to enrich for membrane proteins from human brain tissue. (B) Silver stain showing differential protein banding patterns in each fraction. Immunoblot of PSD-95, a transmembrane domain-containing protein specific to the post synaptic density, demonstrates approximately 2 fold enrichment in the membrane fraction compared with total homogenate. (C) Immunoblots for calnexin, synaptophysin (SYP) and SSBP1 demonstrates that multiple membrane proteins are present in the final membrane fraction.

Figure 2. Human brain membrane factions analyzed by LC-MS/MS

(A) Representative base peak peptide elution profiles of control and AD sample demonstrate the reproducibility between technical replicate 1 (R1) and replicate 2 (R2). (B) Peptide retention time correlation between R1 and R2 from control and AD samples is provided.

Figure 3. Tau enrichment in the membrane fraction of AD cases

An immunoblot (IB) for tau in membrane fraction of individual control and AD cases is shown. Calnexin was used as a loading control. Markers on blots represent molecular weight in kDa.

Figure 4. Confirmation of proteomic changes for UCHL1, Munc-18 and A2M

(A and B) Immunoblot (IB) analyses of the membrane fraction show that UCHL1 (p = 0.0299) and Munc-18 (p = 0.0012) levels were significantly higher in AD cases. (C) Immunoblot analysis of the membrane fraction reveals A2M levels are elevated in 2 of 5 control cases and no AD cases. This difference is not statistically significant (p = 0.15). All immunoblots are representative of three independent experiments. Calnexin was used as a loading control. Human sample information listed in Table 1. Signal intensity was determined by densitometry and statistical analysis was performed using a two-tailed student’s t-test. An asterisk (*) represents significant difference (p < 0.05)