Using differential solubilization and 2‐D gel electrophoresis to visualize increased numbers of proteins in the human cortex and caudate nucleus and putamen

Abstract

The aim of this study was to determine if differential solubilization of human CNS proteins would increase the total number of proteins that could be visualized using 2-D gel electrophoresis. Hence, proteins were solubilized into Tris, CHAPS and SB3-10 before separation across a pH 4-7 IEF gradient and a 12-14% SDS polyacrylamide gel, which could be achieved with a run-to-run variation of 35% in spot intensity. Because Western blot analyses suggested proteins could be in more than one detergent fraction, we completed a conservative analyses of our 2-D gels assuming spots that appeared on multiple gels at the same molecular weight and pI were the same protein.These analyses show that we had visualized over 3000 unique protein spots across three 2-D gels generated from each sample of human frontal cortex and caudate-putamen. This represented, at worst, a significant increase in the number of spots visualized in the acidic protein spectrum compared to what has been reported in other studies of human CNS. This study, therefore, supports the proposal that the analysis of the human CNS proteome using 2-D gel electrophoresis, combined with appropriate sample preparation, can be used to expand the studies on the pathologies of neurological and psychiatric diseases.

Dissecting the Syndrome of Schizophrenia: Progress toward Clinically Useful Biomarkers

“…This approach has led to the visualisation of approximately 3,800 protein spots in the acidic protein fraction from cortex and caudate-putamen, this is close to triple the number of protein spots visualised using crude homogenates as reported in most studies from subjects with Sz [99]. …”

“…At the level of transcriptomics, an increasing knowledge of the complexities of gene expression is producing technologies that can probe mRNA at ever increasing levels of complexity [ 98 ]. In addition, there are efforts to refine existing technologies [ 99 ] and utilise new technologies [ 100 , 101 ] to increase the proportion of the human proteome that can be effectively measured in blood, CNS, or other tissues.…”

“…The capacity to significantly increase the reach of proteomics technologies in psychiatric disease is illustrated by the application of a differential-detergent solubility, multi-gel approach to analysis of the human CNS proteome in tissue from subjects with Sz, BD, and control subjects [ 99 ]. This approach has led to the visualisation of approximately 3,800 protein spots in the acidic protein fraction from cortex and caudate-putamen, this is close to triple the number of protein spots visualised using crude homogenates as reported in most studies from subjects with Sz [ 99 ].…”