Quantitative proteomics of immunocaptured mitochondria from Purkinje Cells

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Mitochondria vary in morphology and function in different tissues, however little is known about their molecular diversity among cell types. To investigate mitochondrial diversity in vivo, we developed an efficient protocol to isolate cell type-specific mitochondria based on a new MitoTag mouse. We profiled the mitochondrial proteome of three major neural cell types in cerebellum and identified a substantial number of differential mitochondrial markers for these cell types in mice and humans. ...

Samples for mass spectrometry were obtained from 8- to 9-week-old, male mice. Mitochondria were immunopurified from cerebellum according to the described protocol with the alteration that the final mitochondrial pellet was washed twice in IB without EDTA and BSA. Sample were lysed in 100 µL SDT lysis buffer (4% w:v SDS, 100 mM DTT in 100 mM Tris-HCl pH 7.6) by heating for 5 min at 95°C and ultrasonication (Vialtweeter: 6 times for 30 s, 100% Amplitude, 50% cycle, max power; Hielscher Ultrasonics). ...