Novel mitochondrial and cytosolic purification pipeline for compartment-specific metabolomics in mammalian disease model tissues.
Metabolomics
; 16(7): 78, 2020 06 23.
Article
en En
| MEDLINE
| ID: mdl-32577914
ABSTRACT
INTRODUCTION:
Mitochondria represent an important milieu for studying the pathogenesis of several major diseases. The need for organelle-level metabolic resolution exists, as mitochondrial/cytosolic metabolites are often diluted beyond detection limits in complex samples. Compartment-specific studies are still hindered by the lack of efficient, cost-effective fractioning methods-applicable to laboratories of all financial/analytical standing.OBJECTIVES:
We established a novel mitochondrial/cytosolic purification pipeline for complimentary GC-TOF-MS and 1H-NMR metabolomics using robust, commercially available fractionation strategies.METHODS:
Magnetic based mitochondria isolation kits (MACS) were adapted for this purpose, accompanied by cytosolic filtering. Yield was assessed through the percentage recovery of citrate synthase (CS; a mitochondrial marker), purity by immunoblotting against compartment-specific proteins and integrity interrogated through the respiratory coupling ratio (RCR). The effects of the kit-based buffers on MS/NMR analyses of pure metabolite standards were evaluated. Finally, biological applicability to mammalian disease models was shown using Ndufs4 mouse brain tissue.RESULTS:
With minor modifications, MACS produced around 60% more mitochondria compared to a differential centrifugation method. Less than 15% of lysosomal LAMP-2 protein was found in the MACS isolates, confirming relative purity-while RCR's above 6 indicate sufficient mitochondrial integrity. The filtering approach effectively depleted mitochondria from the cytosolic fraction, as indicated by negligible Hsp60 and CS levels. Our GC-MS pilot yielded 60-70 features per fraction, while NMR analyses could quantify 6-10 of the most abundant compounds in each fraction.CONCLUSION:
This study provides a simple and flexible solution for mitochondrial and cytosolic metabolomics in animal model tissues, towards large-scale application of such methodologies in disease research.Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Fraccionamiento Celular
/
Citosol
/
Mitocondrias
Límite:
Animals
Idioma:
En
Revista:
Metabolomics
Año:
2020
Tipo del documento:
Article
País de afiliación:
Sudáfrica