Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers.
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 time-of-flight mass spectrometry (MALDI-TOF MS) and were specifically detected by sodium dodecyl sulfate-polyacrylamide 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.
Gubbens
Jacob
J
Department Biochemistry of Membranes, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. j.gubbens@chem.uu.nl
Vader
Pieter
P
Damen
J Mirjam A
JM
O'Flaherty
Martina C
MC
Slijper
Monique
M
de Kruijff
Ben
B
de Kroon
Anton I P M
AI
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D045304:Cytochromes c / Q000032:analysis*; D008928:Mitochondria / Q000737:chemistry / Q000648:ultrastructure; D015394:Molecular Structure; D010743:Phospholipids / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D010743:Phospholipids; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c