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Probing the membrane interface-interacting proteome using photactivatable lipid cross-linkers

By J Gubbens, P. Vader, J.M.A. Damen, M.C. O'Flaherty, M. Slijper, B. de Kruijff and A.I.P.M. de Kroon


To analyze proteins interacting at the membrane interface, a phospholipid analogue was used with a photoactivatable headgroup (ASA-DLPE, N-(4-azidosalicylamidyl)-1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) for selective cross-linking. The peripheral membrane protein cytochrome c from the inner mitochondrial membrane was rendered carbonate wash-resistant by cross-linking to ASA-DLPE in a model membrane system, validating our approach. Cross-link products of cytochrome c and its precursor apocytochrome c were demonstrated by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS) and were specifically detected by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), taking advantage of the intrinsic UV absorbance of the cross-linker. Application of the method to inner mitochondrial membranes from Saccharomyces cerevisae revealed cross-link products of both exogenously added apocytochrome c and endogenous proteins with molecular weights around 34 and 72 kDa. Liquid chromatograpy (LC)-MS/MS was performed to identify these proteins, resulting in a list of candidate proteins potentially cross-linked at the membrane interface. The approach described here provides methodology for capturing phospholipid-protein interactions in their native environment of the biomembrane using modern proteomics techniques

Topics: Farmacie/Biofarmaceutische wetenschappen (FARM), Medical technology, Farmacie(FARM), Biomedische technologie en medicijnen, Pharmacology
Year: 2007
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