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.