Partitioning the Proteome: Phase Separation for Targeted Analysis of Membrane Proteins in Human Post-Mortem Brain

Abstract

Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain.

Omega-3 fatty acid deficiency disrupts endocytosis, neuritogenesis, and mitochondrial protein pathways in the mouse hippocampus

“…Synaptic function was thus significantly implicated as a biological process (endocytosis and exocytosis) and pathway (CME), followed by disturbances in neuritogenesis, mitochondrial dysfunction, and 14-3-3 signaling. As these functions have previously been reported to be abnormal in schizophrenia (Harrison and Weinberger, 2005; English et al, 2011, 2012; Kim et al, 2012; Manji et al, 2012), our data is consistent with the view that n-3 deficiency is an environmental risk factor for schizophrenia (McNamara, 2011). …”

“…This suggestion is in keeping with findings that neuritogenesis (Kawakita et al, 2006; Beltz et al, 2007; He et al, 2009) and mitochondrial function (Eckert et al, 2013) are significantly improved following n-3 augmentation, including that following prenatal stress (Feng et al, 2012), and these changes coincided with improvements in cognitive and depressive-like behaviors. Future studies will focus on understanding the brain proteomic response to n-3 FA supplementation following deficiency, and indeed by studying sub-proteomes such as membrane proteins (English et al, 2012) in n-3 deficient or n-3 ameliorated states.…”

Transformative Impact of Proteomics on Cardiovascular Health and Disease

“…20,41 Protein extraction from cell membranes or the extracellular matrix is difficult because of the hydrophobic nature and poor solubility of these proteins. Poor solubility also limits the utility of 2-dimensional gel electrophoresis for membrane protein analysis.…”

Mind-life continuity: A qualitative study of conscious experience

“…Omics studies began to characterize different biological substrates. Nevertheless, such studies have only recently looked into neuronal structures (English et al, 2012), giving access to solid neuroproteomics databases (Collins et al, 2006;Behan et al, 2009;Hahn et al, 2009;Ishii et al, 2009;Bay es et al, 2011). We propose that omics studies advance towards insights into the biomolecular underpinnings of subjective experience, for instance, the physiological mechanisms involved in strong emotional states (Chiappelli et al, 2006).…”