In addition, they can incur large protein losses and artifactual contamination. A fifth less documented enrichment method, phase separation, is not widely known in proteomics, yet it offers huge potential for routine enrichment and purification of membrane proteins prior to LCMS/MS. Triton X-114 separation was first introduced by Bordier in the early 1980’s and has traditionally been used to enrich for and study membrane proteins in bacteria, although more recently it has been applied to yeast, mouse liver, human cardiac tissue, and porcine brain. To our knowledge, this is the first time phase separation using the Triton X-114 detergent has been applied to human post-mortem brain. Partitioning of the membrane and aqueous proteins is achieved by heating the Triton X-114 to temperatures above 20uC, until it reaches its cloud point. The detergent enters and partitions the lipid bilayer releasing the otherwise insoluble transmembrane proteins. A simple lowspeed centrifugation step recovers the membrane proteins in the detergent phase as an oily pellet, while aqueous proteins are resolved in the supernatant. As protocols with detergent/ membrane combinations have not been well documented or qualified for human brain tissue, the aim of this study was to 1) perform phase separation of detergent and aqueous phase proteins in human post-mortem brain using Triton X-114, and 2) confirm enrichment for membrane proteins in the detergent phase using proteomics. The various proteomic strategies applied in this manuscript are outlined in the study design in Figure 1. Analysis of sub-proteomes and otherwise undetectable protein classes is becoming increasingly important in the field of neuroproteomics, where recent investigations have enriched for and studied the post-synaptic density, lipid rafts, the myelin proteome, neuromelanin granules, and the calmodulin-binding proteome, in human post mortem brain. In this study, we propose a paradigm to enrich for and study membrane proteins in human post-mortem brain. As integral membrane proteins are at the interface between the cell and external environment, and sub-cellular structures, they are important mediators of cell-to –cell signalling, synaptic transmission, cellular transport, and neuroleptic activity. Analysis of this sub-proteome in patients and disease models will greatly aid pathophysiological investigations, yet such studies have not been broadly applied due to the difficulty in recovering and Ponatinib resolving transmembrane proteins. We applied Triton X-114 phase separation to human cortical tissue and confirmed phase separation into DT and AQ phases by comparing the protein banding pattern between control nonenriched tissue and DT and AQ phases. In support, western blotting showed increased expression of transmembrane spanning protein MBP in the DT phase in comparison to the AQ phase, while cytosolic protein GAPDH was depleted in the DT phase and enriched in the AQ phase, as expected. Our LC-MS/MS experiment identified a total of 1154 unique DT phase proteins, and confirmed enrichment where 54% were of membrane protein ontology.