Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study.
One of the most crucial steps in mitochondrial isolation is disruption of intact cells to denude intracellular organelles, but the yield and purity of different disruption protocols have not been well addressed. In the present study, MDCK cells were disrupted by mechanical (sonication and homogenization), physical (repeated freeze/thaw cycles and hypoosmotic burst), and chemical (using Triton X-100, NP-40, or CHAPS) methods. Efficacy of cell disruption was evaluated by trypan blue staining and mitochondria were subsequently isolated by standardized differential centrifugation. The yield of isolation was also determined by measuring protein concentrations, whereas the purity was examined by Janus green B staining, Western blot analyses of markers for mitochondria (COX-4) and other subcellular organelles/locales (i.e., nucleus, cytoplasm, endoplasmic reticulum, and lysosome), transmission electron microscopy, two-dimensional electrophoresis, and Q-TOF MS and/or MS/MS analyses. Our data demonstrated that sonication is the method of choice for disruption of cells prior to mitochondrial isolation for proteome analysis.
Chaiyarit
Sakdithep
S
Medical Proteomics Unit, Office for Research and Development, Department of Immunology and Immunology Graduate Program, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Thongboonkerd
Visith
V
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods; D004285:Dogs; D005615:Freezing; D007668:Kidney / Q000166:cytology; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000032:analysis / Q000378:metabolism; D017830:Octoxynol / Q000378:metabolism; D009994:Osmolar Concentration; D011092:Polyethylene Glycols / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D014465:Ultrasonics / Q000009:adverse effects
D024101:Mitochondrial Proteins; D020543:Proteome; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40