Abstract
Human disease pathophysiology commonly involves metabolic disruption at both the cellular and subcellular levels. Isolated mitochondria are a powerful model for separating global cellular changes from intrinsic mitochondrial alterations. However, common laboratory practices for isolating mitochondria (e.g., differential centrifugation) routinely results in organelle preparations with variable mitochondrial purity. To overcome this issue, we developed a mass spectrometry-based method that quantitatively evaluates sample-specific percent mitochondrial enrichment. Sample-specific mitochondrial enrichment was then used to correct various biochemical readouts of mitochondrial function to a ‘fixed’ amount of mitochondrial protein, thus allowing for intrinsic mitochondrial bioenergetics, relative to the underlying proteome, to be assessed across multiple mouse tissues (e.g., heart, brown adipose, kidney, liver). Our results support the use of mitochondrial-targeted nLC-MS/MS as a method to quantitate mitochondrial enrichment on a per-sample basis, allowing for unbiased comparison of functional parameters between populations of mitochondria isolated from metabolically distinct tissues. This method can easily be applied across multiple experimental settings in which intrinsic shifts in the mitochondrial network are suspected of driving a given physiological or pathophysiological outcome.
Highlights
Human disease pathophysiology commonly involves metabolic disruption at both the cellular and subcellular levels
To determine potential biomarkers of mitochondrial content shared across organs, we isolated mitochondria from several distinct mouse tissues—brown adipose tissue (BAT), heart, kidney, and liver—and subjected them to label-free, quantitative nLC-MS/MS
The purity of each mitochondrial isolation was quantitatively evaluated by comparing the summed abundance of all mitochondrial proteins (i.e., MitoCarta 2.0 positive proteins) to total protein abundance. Total abundance reflects both mitochondrial and non-mitochondrial proteins. This allowed for the generation of a ‘mitochondrial enrichment factor’ (MEF) that reflects mitochondrial protein content per unit of crude isolated mitochondrial protein
Summary
Human disease pathophysiology commonly involves metabolic disruption at both the cellular and subcellular levels. Our results support the use of mitochondrial-targeted nLC-MS/MS as a method to quantitate mitochondrial enrichment on a per-sample basis, allowing for unbiased comparison of functional parameters between populations of mitochondria isolated from metabolically distinct tissues This method can be applied across multiple experimental settings in which intrinsic shifts in the mitochondrial network are suspected of driving a given physiological or pathophysiological outcome. Reflective of this dysregulation, increasing evidence has indicated that mitochondria, the subcellular sites of oxidative phosphorylation (OXPHOS), undergo remodeling in these disease states that may include depressed respiration, increased production of reactive oxygen species, loss of cristae, or loss of mitochondrial v olume[4] These adaptations occur in addition to the broader physiological consequences of altered metabolism such as shifts in protein expression, deposition of lipid droplets, or increased fibrosis. In order to provide experimental contrast, four metabolically diverse tissues were compared: brown adipose tissue, heart, kidney, and liver
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
