{"database":"JPOST Repository","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Mzml":["https://storage.jpostdb.org/JPST000908/files/2020.03.03_C3H_FullFasta.mzML","https://storage.jpostdb.org/JPST000908/files/2020.03.02_C3H_TissueEffect_MitoCarta.mzML"],"Raw":["https://storage.jpostdb.org/JPST000908/files/KFW10.raw","https://storage.jpostdb.org/JPST000908/files/KFW12.raw","https://storage.jpostdb.org/JPST000908/files/KFW6.raw","https://storage.jpostdb.org/JPST000908/files/KFW5.raw","https://storage.jpostdb.org/JPST000908/files/KFW15.raw","https://storage.jpostdb.org/JPST000908/files/KFW13.raw","https://storage.jpostdb.org/JPST000908/files/KFW8.raw","https://storage.jpostdb.org/JPST000908/files/KFW3.raw","https://storage.jpostdb.org/JPST000908/files/KFW1.raw","https://storage.jpostdb.org/JPST000908/files/KFW11.raw","https://storage.jpostdb.org/JPST000908/files/KFW16.raw","https://storage.jpostdb.org/JPST000908/files/KFW4.raw","https://storage.jpostdb.org/JPST000908/files/KFW7.raw","https://storage.jpostdb.org/JPST000908/files/KFW2.raw","https://storage.jpostdb.org/JPST000908/files/KFW9.raw","https://storage.jpostdb.org/JPST000908/files/KFW14.raw"],"Mztab":["https://storage.jpostdb.org/JPST000908/files/2020.03.03_C3H_FullFasta.mzTab","https://storage.jpostdb.org/JPST000908/files/2020.03.02_C3H_TissueEffect_MitoCarta.mzTab"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"omics_type":["Proteomics"],"submitter":["Kelsey Fisher-Wellman"],"species":["Mus Musculus (mouse)"],"full_dataset_link":["https://repository.jpostdb.org/entry/JPST000908"],"submitter_affiliation":["East Carolina Diabetes and Obesity Institute"],"sample_protocol":[""],"repository":["jPOST"],"data_protocol":[""],"pubmed_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."],"pubmed_title":["Novel approach to quantify mitochondrial content and intrinsic bioenergetic efficiency across organs."],"pubmed_authors":["McLaughlin Kelsey L KL, Hagen James T JT, Coalson Hannah S HS, Nelson Margaret A M MAM, Kew Kimberly A KA, Wooten Ashley R AR, Fisher-Wellman Kelsey H KH"],"description_synonyms":["biochemical pathways, MGC130048, Brown Fat, Striadyne, Metabolic Process, Military Uniform, Plantaris, Biological Markers, Viral Marker, Herz, Anterior, Surrogate Endpoints, Activity, determination, Laboratory, Adenosine Triphosphate, Skeletal, Anterior Tibial Muscle, Addresses, Metabolic Concepts, Biochemical, A4, Endpoint, adult heart, protein, Dmef-2, mef2, Autolysosome, Organelle, Serum, Mitochondrial Contractions, composed of, Military Uniforms, TRIM20, Hibernating Gland, Laboratory Markers, diseases, Biological, Concepts, diseases and disorders, Metabolism Concept, protein aggregate, Peroxisome, Phenomenon, imprinted and ancient gene protein, gamma sarcoglycan, strong, C, human disease, Chromium Adenosine Triphosphate, catabolism, proportionality to, Tissue, brown fat, composition, metabolic process resulting in cell growth, purification, cardiac structure, CG1429, free, FMF, Military, MEF2, isolation and purification, ATPsyn b, Immune, Markers, Muscles, DMEF-2, CrATP, scientific observation, Viral Markers, Cr(H2O)4 ATP, sample, Gm9907, gamma-sarcoglycan, biotransformation, Homo sapiens disease, 22.21, SD04091, SIMPLE, DMef-2, Catabolism, Garments, Gastrocnemius, ATP, Chromium Ammonium Salt, Nurse, reniculate kidney, ATPsyn-&bgr, D-Mef2, anatomical protrusion, Viral, Citrate, ATPsyn-b, Surrogate Endpoint, Process, D-MEF2, Krebs cycle, metabolism resulting in cell growth, mef2c, SG-gamma, Mitochondrial Protein, Biochemical Markers, Biologic Marker, not genetically inherited, Autolysosomes, Citric Acid, BC042423, ATP synthase D chain, Voluntary Muscle, Marker, PIG7, Voluntary, mb247, Diseases, Voluntary Muscles, Manganese Salt, Citrate Synthase, Magnesium Salt, secretion, sarcoglycan, simple tissue, cardium, Tricarboxylic Acid Cycle, textus adiposus fuscus, D-mef2, CG11154, AV314029, Brown, Mitochondrial, Epistemology, End Points, Synthase, content, proportionality, School, rate, Immunologic, Krebs, Laboratory Marker, gamma (35kDa dystrophin-associated glycoprotein), experimental procedures, CaATP, disease, Kidneys, DMDA, School Uniforms, spine, Biochemical Marker, 35kD dystrophin-associated glycoprotein, School Uniform, label, Tricarboxylic Acid, Biocatalyst, DmelCG1429, MgATP, Uniforms, Clothes, Fat, E430016J11Rik, DMedf2, other disease, Gastrocnemius Muscle, Lysosome, SGCG_HUMAN, Magnesium Adenosine Triphosphate, experimental, Peptidomics, Clinical Markers, Processes, Biocatalysts, dMef2, dMEF2, Clinical Marker, number, ATP-MgCl2, Gene, branchial heart, Catalyses, Tricarboxylic Acid Cycles, Metabolic Processes, IB, protein-containing complex, mitochondrial, Ximpact, skeletal muscle, presence, TYPE, Adenylpyrophosphate, protrusion, Surrogate End Points, jecur, DAGA4, Surrogate Markers, resilient, isolation, Dmef, Metabolism, Krebs Cycle, tough, ATP-synbeta, Cycle, 35DAG, Mitochondrion, Gene Products, disease or disorder, citric acid cycle, Carrying, MAM, gamma-SG, Metabolism Phenomena, SCG3, dmef2, Cycles, study, Biomarker, Nurse Uniform, proportion, methods, Clinical, DmelCG11154, Tibial Muscle, experimental section, Contraction, Biological Marker, beta-ATPase, ATP MgCl2, Metabolic Concept, BEST:SD04091, Skeletal Muscles, non-neoplastic, Skeletal Muscle, Enzyme, Immunologic Markers, Chromium Salt, Plantaris Muscle, ATPasebeta, DMef2, DMEF2, MnATP, ATPase beta, disorder, Anterior Tibial, Immunologic Marker, ATPB, Citric Acid Cycles, Biologic, TP53I7, striated muscle, measuring, Citrate oxaloacetate-lyase ((pro-3S)-CH2COO(-)--acetyl-CoA), degradation, 35 kDa dystrophin-associated glycoprotein, Uniform, protein complex, MEF-2, Proteins, Serum Markers, disorders, ELFR, End Point, Magnesium Chloride, medical condition, function, Hearts, compositionality, Dmef2, Muscle, Adenosine 5'-(tetrahydrogen triphosphate), impact-a, SGCG, LGMD2C, Immune Marker, Concept, Metabolic Phenomena, ATP-syn-B, Soleus, Metabolism Concepts, Contractions, somatic muscle, count in organism, Atriphos, Brown Adipose Tissue, Experiment, Nurse Uniforms, ML-1, native protein, oxidative TCA cycle, Surrogate End Point, Protein, chemical analysis, Phenomena, MEF, Mef, condition, imprinted and ancient gene protein homolog, IMPACT, ENSMUSG00000053512, metabolism, Metabolic Phenomenon, Mef-2, Biologic Markers, multicellular organism metabolic process, Serum Marker, Manganese Adenosine Triphosphate, biodegradation, DMDA1, Soleus Muscle, Metabolic, Garment, Surrogate, mef, Endpoints, mef-2, sample population, Surrogate Marker, Brown Adipose, Mitochondrial Contraction, Protein Gene Products, iecur., Gene Proteins, Livers, mitochondria, TCA cycle, l(2)46CFr, Isolation of Nuclei TAgged in specific Cell Types, ATPIB, SCARMD2, structure, quotient, assay, BAT, General activity, RWDD5, Calcium Salt, Anabolism, skeletal muscle system, Immune Markers"],"pubmed_abstract_synonyms":["other disease, BODYFAT, Herz, Procedures, experimental, Laboratory, Mus domesticus, adult heart, branchial heart, Gm695, Organelle, Spectrum Analyses, fat, Mitochondrial Contractions, mitochondrial, House Mouse, jecur, method, Techniques, %, diseases, House, Method, method used in an experiment, Mitochondrion, Mass, Studies, disease or disorder, Mus musculus domesticus, diseases and disorders, Analysis, fatty tissue, Mice, Technique, fat tissue, Mass Spectroscopy, Mass Spectrum Analysis, human disease, methods, Analyses, adipose system, Swiss, MS2, experimental section, Contraction, dysfunction, Tissue, iecur, Swiss Mice, cardiac structure, non-neoplastic, Study, b, Methodological Studies, sample, TRP-1, bodyfat, disorder, Homo sapiens disease, house mouse, isa, reniculate kidney, Tyrp, Proteins, disorders, mouse, total expressed protein, Mitochondrial Protein, medical condition, function, Hearts, Procedure, Spectrum Analysis, results, Spectroscopy, Contractions, Body Fat, MS, ATP synthase D chain, Mus, brown, adp, Protein, condition, laboratory, cardium, fixed, Mass Spectrum Analyses, tandem MS, Mass Spectrum, Mus musculus, Oca3, Mitochondrial, distinct, MS/MS, mice, Swiss Mouse, Spectrometry, common, House Mice, pathophysiology, Methodological, Methodological Study, sample population, domesticus, Laboratory Mice, experimental procedures, Mitochondrial Contraction, plan specification, Livers, disease, Kidneys, mitochondria, fatty depot, adipose, Mouse, mitochondrial., variable, TRP1, Proteomes, Laboratory Mouse"],"pubmed_title_synonyms":["Mitochondrial, composition, ATP synthase D chain, compositionality, mitochondrial, composed of, structure, content, Productivity."],"name_synonyms":["Mass Spectrum Analysis, Mass Spectrum, Mus musculus, Laboratory Mice., Mitochondrial, Analyses, Laboratory, Swiss, mice, content, Mus domesticus, number, mouse, Swiss Mouse, Spectrometry, total expressed protein, composition, House Mice, Swiss Mice, Spectrum Analyses, compositionality, mitochondrial, composed of, Spectrum Analysis, presence, House Mouse, domesticus, Productivity, Spectroscopy, count in organism, MS, ATP synthase D chain, House, Mus, structure, Mass, Mus musculus domesticus, Mouse, Analysis, house mouse, Mice, Proteomes, Laboratory Mouse, Mass Spectrum Analyses, Mass Spectroscopy"],"additional_accession":[]},"is_claimable":false,"name":"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. \nOne 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. \nThe 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 \ncomprehensive 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. ","dates":{"publication":"Fri Jul 09 00:00:00 BST 2021"},"accession":"PXD020280","cross_references":{"TAXONOMY":["10090"],"pubmed":["33077793"]}}