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 m...

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Bibliographic Details
Published in:Journal of proteome research 2007-05, Vol.6 (5), p.1951-1962
Main Authors: Gubbens, Jacob, Vader, Pieter, Damen, J. Mirjam A, O'Flaherty, Martina C, Slijper, Monique, de Kruijff, Ben, de Kroon, Anton I. P. M
Format: Article
Language:English
Subjects:
Animals
Cell Membrane - chemistry
Chromatography, Liquid
Cross-Linking Reagents - chemistry
Cytochromes c - analysis
Mitochondria - chemistry
Mitochondria - ultrastructure
Molecular Structure
Phospholipids - chemistry
Proteome - analysis
Saccharomyces cerevisiae - chemistry
Saccharomyces cerevisiae - cytology
Saccharomyces cerevisiae Proteins - analysis
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Tandem Mass Spectrometry
Ultraviolet Rays
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Summary: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. Keywords: Photolabeling • ASA-PE • Saccharomyces cerevisiae • mitochondria • cytochrome c • biomembranes • proteomics • lipid−protein interactions
ISSN:1535-3893
1535-3907
DOI:10.1021/pr060561a