Proteome profile of functional mitochondria from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS

Abstract

Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioenergetic function in vitro. The purpose of this study was to characterize the composition of such preparations using a proteomics approach. Mitochondria isolated from human vastus lateralis biopsies were functional as evidenced by their response to carbohydrate and fat-derived fuels. Using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were detected, and 487 of these were assigned to the mitochondrion, including the newly characterized SIRT5, MitoNEET and RDH13. Proteins detected included 9 of the 13 mitochondrial DNA-encoded proteins and 86 of 104 electron transport chain (ETC) and ETC-related proteins. In addition, 59 of 78 proteins of the 55S mitoribosome, several TIM and TOM proteins and cell death proteins were present. This study presents an efficient method for future qualitative assessments of proteins from functional isolated mitochondria from small samples of healthy and diseased skeletal muscle.

Diversity of human skeletal muscle in health and disease: Contribution of proteomics

“…Special emphasis shall also be placed on the study of human skeletal muscle mitochondria for the possible assessment of proteins from small samples of healthy and diseased skeletal muscles [37,38]. In the above investigation, 100 mg of tissue biopsies were utilized for mitochondrial enrichment.…”

Mitochondrial Proteomics: From Structure to Function

“…Up-to-date gel-free proteomic techniques, such as LC/LC-MSMS (Reinders & Sickmann, 2007;Lefort et al, 2009) have complemented gel-based experiments, leading mitochondrial molecular investigation to higher levels of data production and accuracy. It is well known that protein quantitation and low abundant protein detection have been among the major limitations on proteomic research.…”

“…Therefore the cataloging of the entire organelle proteome is of inestimable value to biomedical research, and further understanding of molecular mitochondrial modulation of positive stimulus or pathological insults shows enormous pharmacological potential (Fearnley et al, 2007;Wang et al, 2009). By different proteomic methods, much progress has been made in this direction in order to target and catalogue mitochondrial proteins from different organisms, such as humans (Rabilloud et al, 1998;Lefort et al, 2009), rodents Doran et al, 2009), fungi (Grinyer et al, 2004) and plants (Taylor et al, 2011). In addition to simple proteome cataloging, several studies have been carried out to compare mitochondrial proteomes from different tissues (Forner et al, 2006;Fang & Lee, 2009;Forner et al, 2009), as well as from organ structures (e.g.…”

Evaluation of Mitochondrial Functions and Dysfunctions in Muscle Biopsy Samples

“…Recently, a proteomic study of mitochondrial proteins (mitoproteome) reported that more than 80% of ETC proteins can be identified (Lefort et al, 2009). The authors were able to reliably detect proteins implicated in ROS generation, scavenging, FA oxidation and apoptosis.…”