Cloud-Point Extraction and Delipidation of Porcine Brain Proteins in Combination with Bottom-Up Mass Spectrometry Approaches for Proteome Analysis

Abstract

In this study, temperature-induced phase fractionation also known as cloud-point extraction (CPE) with the nonionic surfactant Triton X-114 was used to simultaneously extract hydrophobic and hydrophilic proteins from porcine brain tissue. Various protein precipitation/delipidation procedures were investigated to efficiently remove lipids and detergents while retaining maximum protein recoveries. The best performing delipidation method was then used in combination with CPE to compare three different mass spectrometry (MS) based "bottom-up" proteomic approaches for protein analysis of the porcine brain. In the first approach, the intact proteins were initially separated by one-dimensional (1D) gel electrophoresis. The excised protein bands were digested with trypsin, and the peptides were separated by reversed phase nanoliquid chromatography (RP-nanoLC) followed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) analysis. The other bottom-up proteomic approaches were based on first enzymatical digestion of the proteins followed by RP-nanoLC separation in combination with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) or on the combination of in-solution isoelectric focusing (IEF) with ESI-nanoLC-MS/MS of the IEF separated peptides. In total, we found and unambiguously identified 331 unique proteins. The overlap between different techniques was about 10%, showing that the use of multiple proteomic approaches is beneficial to yield a better coverage of the proteome. Furthermore, the overlap between the CPE extracted hydrophilic and hydrophobic proteins was rather small (9-16%), indicating an efficient sample preparation technique to extract and separate hydrophilic and hydrophobic proteins from brain tissue. The percentage of identified membrane proteins was 27%, which is in accordance to the fact that about one-third of all genes in various organisms encode for this class of proteins. The results indicate that cloud point extraction is a promising sample preparation tool, which allows simultaneous in depth studies of brain derived membrane proteins as well as hydrophilic proteins. This technique can be very useful when studying human central nervous system (CNS) tissue or animal models of neurological diseases.