A novel subfractionation approach for mitochondrial proteins: A three-dimensional mitochondrial proteome map

Abstract

As mitochondria play critical roles in both cell life and cell death, there is great interest in obtaining a human mitochondrial proteome map. Such a map could potentially be useful in diagnosing diseases, identifying targets for drug therapy, and in screening for unwanted drug side effects. In this paper, we present a novel approach to obtaining a human mitochondrial proteome map that combines sucrose gradient centrifugation with standard two-dimensional gel electrophoresis. The resulting three-dimensional separation of proteins allows us to address some of the problems encountered during previous attempts to obtain mitochondrial proteome maps such as resolution of proteins and solubility of hydrophobic proteins during isoelectric focusing. In addition, we show that this new approach provides functional information about protein complexes within the organelle that is not obtained with two-dimensional gel electrophoresis of whole mitochondria.

Age-dependent changes in myocardial matrix metalloproteinase/tissue inhibitor of metalloproteinase profiles and fibroblast function

“…To extract easily soluble proteins (e.g., cytoplasmic proteins), the LV half was homogenized in soluble lysis buffer (0.25 M sucrose and 1 mM EDTA) and centrifuged at 13,000 Âg for 10 min [19]. To extract insoluble proteins (e.g., membrane proteins), the insoluble pellet was resuspended in chaotropic membrane extraction reagent (7 M urea, 2 M thiourea, and the detergent amidosulfobetaine-14; Sigma).…”

An improved formulation of SYPRO Ruby protein gel stain: Comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

“…As is standard with SDS-PAGE, the stacking gel buffer concentration was 0.125 M Tris-HCl, pH 6.8, the separating gel buffer concentration was 0.375 M Tris-HCl, pH 8.8 and the reservoir buffer concentration was 0.025 M Tris, 0.192 M glycine, pH 8.3. For 2-DE, bovine heart mitochondria were isolated as previously described [24]. Approximately 150 mg protein was applied to each pH 3-10 Immobiline DryStrip immobilized pH gradient gel (Amersham Pharmacia Biotech, Piscataway, NJ, USA) by the rehydration method and large format 2-DE gel electrophoresis was performed using an Investigator system by manufacturer's instructions (Genomic Solutions, Ann Arbor, MI, USA).…”

High throughput two-dimensional blue-native electrophoresis: A tool for functional proteomics of mitochondria and signaling complexes

“…First, many important mitochondrial proteins such as the complexes of the respiratory chain are hydrophobic membrane proteins, and will precipitate at the basic pole during IEF [11][12][13]. Second, all functional information regarding protein:protein interactions is lost due to the high stringency, denaturing conditions of both gel dimensions.…”

“…In this respect, Hanson et al [13] recently proposed a 3-D method in which high-resolution sucrose density gradients are used to separate functional protein complexes prior to conventional IEF SDS-PAGE 2-D gels. However, the requirement for large amounts of mitochondrial protein (1-5 mg) and an overnight centrifugation step may limit the application of this technique for high throughput analysis of samples where material is limited, as frequently occurs with experiments using cell culture.…”