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...
| Main Authors: | , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
2007
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| _version_ | 1797102130716213248 |
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| author | Gubbens, J Vader, P Damen, J O'Flaherty, M Slijper, M de Kruijff, B de Kroon, A |
| author_facet | Gubbens, J Vader, P Damen, J O'Flaherty, M Slijper, M de Kruijff, B de Kroon, A |
| author_sort | Gubbens, J |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T06:01:34Z |
| format | Journal article |
| id | oxford-uuid:ec5d0ba2-f372-4f3e-9008-57eeaf3f7123 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T06:01:34Z |
| publishDate | 2007 |
| record_format | dspace |
| spelling | oxford-uuid:ec5d0ba2-f372-4f3e-9008-57eeaf3f71232022-03-27T11:16:57ZProbing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:ec5d0ba2-f372-4f3e-9008-57eeaf3f7123EnglishSymplectic Elements at Oxford2007Gubbens, JVader, PDamen, JO'Flaherty, MSlijper, Mde Kruijff, Bde Kroon, ATo 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. |
| spellingShingle | Gubbens, J Vader, P Damen, J O'Flaherty, M Slijper, M de Kruijff, B de Kroon, A Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers. |
| title | Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers. |
| title_full | Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers. |
| title_fullStr | Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers. |
| title_full_unstemmed | Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers. |
| title_short | Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers. |
| title_sort | probing the membrane interface interacting proteome using photoactivatable lipid cross linkers |
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