| Alteration of the mitochondrial proteome and altered mitochondrial function has been implicated in a variety of degenerative diseases, heart disease, aging and cancer. Based upon the human genome there is estimated to be between 1000--2000 proteins constituting the mitochondrial proteome. Two-dimensional gel electrophoresis (2-DE) has the potential to resolve up to 10,000 proteins in a single gel, however just over 600 mitochondrial proteins have been identified at the molecular level. The hydrophobic nature of membrane proteins and the basic isoelectric point of many mitochondrial inner membrane proteins makes this subproteome particularly difficult to resolve by 2-DE. In this body of work a highly purified mitochondrial inner membrane subproteome was analyzed utilizing the resolving power of 2-DE with pH 4--7, 3--10, and 6--11 immobilized pH gradients. A database of over 100 proteins, 20 of which were post translationally modified, was created of the proteins associated with and spanning the mitochondrial inner membrane. Extensive optimization was required to improve protein solubility prior to and during resolution by isoelectric focusing. Nearly every protein identified by 2-DE was of mitochondrial origin, demonstrating the high degree of purity of the inner membrane subproteome fractionation. To supplement the 2-DE database a subfractionation enriched for mitochondrial inner transmembrane proteins was prepared and resolved by 1-D SDS-PAGE. By this method, 40 transmembrane containing proteins from the mitochondrial inner membrane, outer membrane, and endoplasmic reticulum were identified. This represents the development of a method for the proteomic analysis of mitochondrial inner membrane proteins that can subsequently be applied to various models. |
