Organelle proteomics: reduction of sample complexity by enzymatic in-gel selection of native proteins

Abstract

One major problem in proteomics is the biochemical complexity of living cells. Therefore, strategies are needed to reduce the number of proteins to a manageable amount, enabling researchers to make a statement concerning protein functions. One possibility is the isolation of organelles, which reduces the protein complexity, e.g., for the chloroplast to an estimated number of 2,700 different proteins. For further limitation of the protein number, proteins can be divided into membrane and soluble proteins, which can be analyzed separately in a subsequent step. For membrane proteins, blue native polyacrylamide gel electrophoresis (BN-PAGE) in combination with enzymatic in-gel assays (e.g. detection of NADPH dehydrogenases) is a suitable method for a fast and easy visualization and identification of only one class of membrane proteins.