MitoPlex: A Targeted Multiple Reaction Monitoring Assay for Quantification of a Curated Set of Mitochondrial Proteins
- Organism: Mus musculus
- Instrument: QTRAP 6500+,TripleTOF 5600+
Mitoplex, MRM, metabolomics, statin myopathy, striated muscle, mitochondrial function
- Lab head:
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.
Targeted MRM Assays (Tier 2) were created to quantify up to 32 mitochondrial proteins selected to be informative of major functional pathways (TCA cycle, ETC, mitochondrial remodeling, etc). A concurrent protein and metabolite extraction protocol was developed to enable analysis of both analyte types from the same cell or tissue sample. A targeted metabolomic panel was measured concomitantly with mitochondrial proteins for comparison. The performance and biological informativeness of the assay was assessed in a series of experiments interrogating metabolic differences between differentiating C2C12 skeletal muscle cell in vitro model and between striated muscle types (oxidative and glycolytic skeletal muscle, cardiac muscle) with, and without, exposure to statins.
C2C12 myoblast and myotube cultures
Mouse soleus muscle
Mouse gastrocnemius muscle
Mouse cardiac muscle
Created on 3/4/20, 2:59 PM