Abstract
A new type of technology in proteomics was developed in order to separate a complex protein mixture and analyze protein functions systematically. The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification. In order to demonstrate the feasibility of this functional proteomics approach, NADH and NADPH-dependent oxidases, major redox enzyme families, were identified from mice cochlear tissue after a specific drug treatment. By comparing the enzymatic activity between mice that were treated with a drug and a control group significant changes were observed. Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment. In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment.
Highlights
Proteins play essential roles in numerous biological processes
For specific fractions with an enzymatic activity of interest, mass spectrometry was used for further protein identification. This current study focused on oxidative enzymes, especially NADH and NADPH-dependent oxidases because in the cochlea, several NADH and NADPH-dependent oxidases have been implicated in hearing disorders such as age-related and noise-induced hearing loss [15]
No reducing reagent was used in the samples during Isoelectric focusing (IEF) to keep the disulfide bonds intact in proteins
Summary
Proteins play essential roles in numerous biological processes. Defining their roles and understanding their interaction networks are important in the discovery of basic biological processes and elucidation of important disease mechanisms. Over 20% of human genes code for enzymes which are responsible for most cellular biochemical processes [1]. Despite their importance, only a limited portion of the enzymes in the human genome have been characterized. Traditional studies of enzymes were carried out on either individual proteins or a small number of proteins which limits the ability to systematically understand the roles of these proteins in various biological functions. Gene expressions can be analyzed with a gene chip or other high
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