This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Perturbations in skeletal muscle mitochondrial (mt) protein expression may contribute to the pathogenesis of metabolic disorders in the elderly. Skeletal muscle is the largest, mt-rich tissue in the body and the primary site for glucose storage. Normal muscle mt protein expression is required for normal glucose and fatty acid metabolism. However, we lack sensitive, specific and comprehensive analytical tools for examining the human muscle mt proteome. We propose to develop sensitive, mt-specific, mass spectrometry (MS)-based comparative proteomics tools and approaches that can be used to identify, characterize and quantify the human muscle mt proteome in small muscle samples (10-100mg) obtained from genetically modified mice, and well-characterized young (18-35 yr) and elderly men and women (65-80yr) with normal or impaired glucose tolerance. We hypothesize that novel strategies will provide more comprehensive organelle-specific coverage of the muscle mt proteome, and allow for more simplified, comparative (young vs old), and interpretable outcomes for characterizing alterations in low abundance muscle mt proteins in young vs elderly adults, and genetically modified vs wild-type mice. We hypothesize that by targeting muscle mt proteins and by employing MS techniques to detect many mt proteins in a single muscle sample, we will identify and characterize global alterations in muscle mt protein expression and post-translational modifications that are associated with aging and insulin resistance. Specifically, we will extract and separate muscle mt proteins using customized sub-cellular fractionation, protein enrichment and immunoprecipitation. We will identify and characterize muscle mt proteins/peptides using 2D-differential fluorescence gel electrophoresis, 1D-and 2D-liquid chromatographic separation, and isotope coded affinity tagging, each followed by MALDI-TOF-TOF-MS and nano-LC-FT-tandem MS for accurate mass measurements and amino acid sequencing. We will discover new and important mt protein forms, and generate novel approaches and new hypotheses about the pathogenesis of muscle mt-based metabolic disorders in the elderly. These strategies have been used to examine proteomes in small organisms, but they need to be applied to complex human tissues (muscle) involved in disorders of human substrate metabolism, and that might ultimately lead to novel treatments. To do this, we will take advantage of the expertise and MS instrumentation available at Washington University.
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