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Phosphoproteome Analysis of Functional Mitochondria Isolated from Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes*

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Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.

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*

This work was supported by the Graduate School for Molecular Metabolism at the University of Southern Denmark (to X. Z.), The Novo Nordisk Foundation (Excellence Project 2009), The Danish Medical Research Council (to K. H.), The Danish Research Agency, and the Lundbeck Foundation (to O. N. J.).

This article contains supplemental Figs. 1–4 and Tables 1–5.

1

The abbreviations used are:

    PTM

    post-translational modification

    TiO2

    titanium dioxide

    ZIC-HILIC

    zwitterionic hydrophilic interaction chromatography

    CPP

    calcium phosphate precipitation

    LTQ

    linear ion trap

    MSA

    multistage activation

    FA

    formic acid

    IPI

    International Protein Index

    VDAC

    voltage-dependent anion channel

    CKII

    casein kinase II

    DNAPK

    DNA-dependent protein kinase.