Dataset Information

MitoPlex: A Targeted Multiple Reaction Monitoring Assay for Quantification of a Curated Set of Mitochondrial Proteins

ABSTRACT: Mitochondria are the major source of cellular energy (ATP), as well as critical mediators of widespread functions such as cellular redox balance, apoptosis, and metabolic flux. Methods to quantify mitochondrial content are limited to low throughput immunoassays, measurement of mitochondrial DNA, or relative quantification by untargeted mass spectrometry. Here, we present a high throughput, focused multiple reaction monitoring based assay of 32 proteins critical to central carbon chain metabolism and overall mitochondrial function termed ‘MitoPlex’. We coupled this protein multiplex with a parallel analysis of 218 metabolites extracted in tandem from the same sample. In tests of its biological applicability, “MitoPlex plus metabolites” indicated profound effects of HMG-CoA Reductase inhibition (e.g., statin treatment) on mitochondria of differentiating C2C12 skeletal myoblasts, as well as a clear opposite trend of statins to promote mitochondrial protein expression and metabolism in heart and liver, while suppressing mitochondrial protein and aspects of metabolism in the skeletal muscle of C57Bl6 mice. Our results not only reveal new insights into the metabolic effect of statins in skeletal muscle, but present a new high throughput, reliable MS-based tool to study mitochondrial dynamics in both cell culture and in vivo models.

ORGANISM(S): Mus Musculus

SUBMITTER: Sarah Parker

PROVIDER: PXD017884 | panorama | Thu Apr 09 00:00:00 BST 2020

REPOSITORIES: PanoramaPublic

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MitoPlex: A targeted multiple reaction monitoring assay for quantification of a curated set of mitochondrial proteins.

Stotland Aleksandr B AB Spivia Weston W Orosco Amanda A Andres Allen M AM Gottlieb Roberta A RA Van Eyk Jennifer E JE Parker Sarah J SJ

Journal of molecular and cellular cardiology 20200329

Mitochondria are the major source of cellular energy (ATP), as well as critical mediators of widespread functions such as cellular redox balance, apoptosis, and metabolic flux. The organelles play an especially important role in the maintenance of cardiac homeostasis; their inability to generate ATP following impairment due to ischemic damage has been directly linked to organ failure. Methods to quantify mitochondrial content are limited to low throughput immunoassays, measurement of mitochondri ...[more]

PMID: 32234390

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Project description:Recent studies on mouse and human skeletal muscle (SM) demonstrated the important link between mitochondrial function and the cellular metabolic adaptation. To identify key compensatory molecular mechanisms in response to chronic mitochondrial distress, we analyzed mice with ectopic SM respiratory uncoupling in uncoupling protein 1 transgenic (UCP1-TG) mice as model of muscle-specific compromised mitochondrial function. Here we describe a detailed metabolic reprogramming profile associated with mitochondrial perturbations in SM, triggering an increased protein turnover and amino acid metabolism with induced biosynthetic serine/1-carbon/glycine pathway and the longevity-promoting polyamine spermidine as well as the trans-sulfuration pathway. This is related to an induction of NADPH-generating pathways and glutathione metabolism as an adaptive mitohormetic response and defense against increased oxidative stress. Strikingly, consistent muscle retrograde signaling profiles were observed in acute stress states such as muscle cell starvation and lipid overload, muscle regeneration, and heart muscle inflammation, but not in response to exercise. We provide conclusive evidence for a key compensatory stress-signaling network that preserves cellular function, oxidative stress tolerance, and survival during conditions of increased SM mitochondrial distress, a metabolic reprogramming profile so far only demonstrated for cancer cells and heart muscle.-Ost, M., Keipert, S., van Schothorst, E. M., Donner, V., van der Stelt, I., Kipp, A. P., Petzke, K.-J., Jove, M., Pamplona, R., Portero-Otin, M., Keijer, J., and Klaus, S. Muscle mitohormesis promotes cellular survival via serine/glycine pathway flux. Control C57BL/6J wildtype male mice, aged 12 weeks, are compared to UCP1 transgene littermates fed purified low fat diet (BIOCLAIMS, Hoevenaars et al., Genes and Nutrition 2012) for 12 weeks . At the end, mice were sacrificed, gastrocnemius skeletal muscle was immediately dissected and snap frozen in liquid nitrogen. Total RNA was isolated, quantified and qualified, and subsequently used for global gene expression profiling using Agilent 8x60K microarrays. In total, 6 arrays of individual WT and 7 arrays of individual UCP1-TG samples were normalized, together with samples from the complete SUPERSERIES.

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Dataset Information

MitoPlex: A Targeted Multiple Reaction Monitoring Assay for Quantification of a Curated Set of Mitochondrial Proteins

Publications

MitoPlex: A targeted multiple reaction monitoring assay for quantification of a curated set of mitochondrial proteins.

Similar Datasets

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