Protocol to characterize mitochondrial supercomplexes from mouse tissues by combining BN-PAGE and MS-based proteomics

• Published in: STAR protocols Vol. 3; no. 1; p. 101135
• Main Authors: Moreno-Justicia, Roger, Gonzalez-Franquesa, Alba, Stocks, Ben, Deshmukh, Atul S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.03.2022; Elsevier
• Subjects: Animals; Electrophoresis; Mass Spectrometry; Metabolism; Mice; Mitochondria - chemistry; Protein Biochemistry; Proteomics; Proteomics - methods; Protocol; Protein Biochemistry; Metabolism; Mass Spectrometry; Proteomics
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101135

The assembly of mitochondrial respiratory complexes into supercomplexes has significant implications for mitochondrial function. This protocol details mitochondrial isolation from mouse tissues and the use of blue native gel electrophoresis (BN-PAGE) to separate pre-identified mitochondrial supercomplexes into different gel bands. We then describe the excision of the individual bands, followed by in-gel protein digestion and peptide desalting for mass spectrometry (MS)-based proteomics. This protocol provides a time-efficient measurement of the abundance and distribution of proteins within known supercomplexes. For complete details on the use and execution of this profile, please refer to Gonzalez-Franquesa et al. (2021). [Display omitted] •Protocol to study mitochondrial supercomplexes composition from mouse tissue•MS-based proteomics of visible mitochondrial supercomplexes separated on BN-PAGE•Measurement of the abundance and distribution of proteins within known supercomplexes•This protocol can be applied to protein complexes visible by Coomassie blue staining The assembly of mitochondrial respiratory complexes into supercomplexes has significant implications for mitochondrial function. This protocol details mitochondrial isolation from mouse tissues and the use of blue native gel electrophoresis (BN-PAGE) to separate pre-identified mitochondrial supercomplexes into different gel bands. We then describe the excision of the individual bands, followed by in-gel protein digestion and peptide desalting for mass spectrometry (MS)-based proteomics. This protocol provides a time-efficient measurement of the abundance and distribution of proteins within known supercomplexes.