PXD020280

PXD020280 is an original dataset announced via ProteomeXchange.

Title	Mitochondrial content normalization using mass spectrometry allows quantitative evaluation of intrinsic bioenergetic efficiency and the underlying proteome across mouse organs.
Description	To separate global changes to metabolism from intrinsic mitochondrial remodeling, investigators commonly use isolated mitochondrial preparations obtained through differential centrifugation. This simple process has been reliably implemented in a variety of tissues to produce intact, functional mitochondria for bioenergetic evaluation. For the normalization of data between isolations from the same tissue type, values are typically scaled to the amount of protein used per experiment. However, the crude mitochondrial pellet acquired through differential centrifugation will also contain non-mitochondrial contaminants including lysosomes, peroxisomes, and portions of other subcellular organelles that are of similar density to mitochondria. Further, it has been shown that contamination is not uniform across tissues, as each tissue maintains a different proportion of these organelles to perform its specialized functions. Similarly, given the cellular consequences of disease listed above, there will likely be an additional impact of disease state upon the purity of the mitochondrial preparation. Accordingly, equitable comparison of mitochondrial function across different tissues, as well as between diseased/healthy states, requires reliable normalization that corrects for the mitochondrial purity across subcellular isolations. One prevailing strategy for normalization is to estimate mitochondrial content through measuring the activity of citrate synthase (CS), an enzyme at the intersection of fuel catalysis and entry of metabolites into the citric acid cycle. Several groups have reported a strong correlation between mitochondrial content and CS activity in skeletal muscle. However, to our knowledge, this correlation has not been validated in other tissues, nor in different disease states, potentially limiting its application for evaluating isolation purity for all experimental models. Moreover, as the mitochondrion represents a complex collection of integrated pathways, it seems unlikely that the activity of any single enzyme would be reflective of mitochondrial content across tissues with differing energetic demands or constraints. The present study sought to address this technical barrier inherent to quantifying inter-mitochondrial differences across organs through the use of label-free, mitochondrial-targeted nanoLC-MS/MS paired with comprehensive bioenergetic phenotyping. By carrying out quantitative proteomics screens on aliquots of mitochondria used for functional analysis, this allowed us to directly compute mitochondrial vs. non-mitochondrial protein on a per sample basis. Such analyses generated a mitochondrial enrichment factor (MEF) that empirically reflected the mitochondrial purity of a given isolation. We subsequently used this MEF to identify potential protein biomarkers of mitochondrial content shared across tissues, as well as directly compare mitochondrial bioenergetic fluxes between tissues through differential protein expression, high resolution respirometry, and ATP production profiles. In order to provide experimental contrast, we chose to compare four metabolically diverse tissues: brown adipose tissue, heart, kidney, and liver.
HostingRepository	jPOST
AnnounceDate	2021-07-09
AnnouncementXML	Submission_2022-09-18_03:03:32.969.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Kelsey Fisher-Wellman
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; TMT6plex reporter+balance reagent N-acylated residue; TMT6plex reporter+balance reagent acylated N-terminal
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2020-07-09 13:00:03	ID requested
1	2021-07-08 08:00:05	announced
2	2022-04-21 10:10:15	announced	2022-04-22: Updated PubMed.
⏵ 3	2022-09-18 03:03:33	announced	2022-09-18: Updated FTP location.

McLaughlin KL, Hagen JT, Coalson HS, Nelson MAM, Kew KA, Wooten AR, Fisher-Wellman KH, Novel approach to quantify mitochondrial content and intrinsic bioenergetic efficiency across organs. Sci Rep, 10(1):17599(2020) [pubmed]

submitter keyword: proteomics, bioenergetics, mitochondria, efficiency, metabolism, respirometry

Kelsey Fisher-Wellman
lab head
Kelsey Fisher-Wellman
contact affiliation	East Carolina Diabetes and Obesity Institute
dataset submitter

jPOST dataset URI

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