title: Binding mitochondria to cryogel monoliths allows detection of proteins specifically released following permeability transition.
abstract: Following proapoptotic signals such as calcium-induced mitochondrial permeability transition or translocation of proapoptotic proteins, mitochondria induce cell death through release of apoptogenic proteins. The mechanism of release and the identity of the released proteins are currently debated. Earlier attempts at identification of the apoptogenic proteins have been hampered by a high nonspecific background. Our aim was to develop a novel method where background release was eliminated, allowing proteins specifically released from mitochondria following proapoptotic stimulation to be identified. Liver mitochondria were immobilized and washed on cryogel monoliths prior to induction of protein release (calcium or Bid/Bax). Immobilized mitochondria exhibited normal morphology and swelling response and retained respiratory activity. The released proteins were collected, concentrated, separated on polyacrylamide gels which were cut into pieces, trypsin-digested, and analyzed using liquid chromatography-tandem mass spectrometry. Control samples contained no protein, and stimulation with calcium and Bid/Bax resulted in identification of 68 and 82 proteins, respectively. We conclude that, in combination with the robust proteomic approach, immobilization on cryogel monoliths is a fruitful approach for studying specific protein release from isolated mitochondria. We propose that this method is a powerful tool to further characterize the role of mitochondria in cell death induction.
authors.affiliation: Laboratory for Experimental Brain Research, Wallenberg Neuroscience Center, Lund University, 221 84 Lund, Sweden. maria.teilum@med.lu.se
authors.forename: M
authors.initials: M
authors.lastname: Teilum
authors.forename: M J
authors.initials: MJ
authors.lastname: Hansson
authors.forename: M B
authors.initials: MB
authors.lastname: Dainiak
authors.forename: R
authors.initials: R
authors.lastname: Månsson
authors.forename: S
authors.initials: S
authors.lastname: Surve
authors.forename: E
authors.initials: E
authors.lastname: Elmér
authors.forename: P
authors.initials: P
authors.lastname: Onnerfjord
authors.forename: G
authors.initials: G
authors.lastname: Mattiasson
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000494:pharmacology; D001681:Biological Assay; D001798:Blood Proteins / Q000737:chemistry* / Q000648:ultrastructure; D002118:Calcium / Q000494:pharmacology; D059985:Cryogels; D005353:Fibronectins / Q000737:chemistry* / Q000648:ultrastructure; D020100:Hydrogels; D046529:Microscopy, Electron, Transmission; D008930:Mitochondria, Liver / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D051028:bcl-2-Associated X Protein / Q000494:pharmacology
chemical_list: D051023:BH3 Interacting Domain Death Agonist Protein; D001798:Blood Proteins; D059985:Cryogels; D005353:Fibronectins; D020100:Hydrogels; D011506:Proteins; D051028:bcl-2-Associated X Protein; D002118:Calcium