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Related Experiment Video

Updated: Jul 16, 2026

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
10:49

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Published on: September 20, 2016

Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers.

Jacob Gubbens1, Pieter Vader, J Mirjam A Damen

  • 1Department 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

Journal of Proteome Research
|March 23, 2007
PubMed
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Researchers developed a new method using a photoactivatable phospholipid analogue (ASA-DLPE) to study membrane protein interactions. This technique successfully identified proteins cross-linked to phospholipids in yeast mitochondria, advancing biomembrane research.

Area of Science:

  • Biochemistry
  • Proteomics
  • Membrane Biology

Background:

  • Understanding protein interactions at the biomembrane interface is crucial for cellular function.
  • Existing methods may not fully capture in vivo phospholipid-protein interactions.
  • Cytochrome c is a key peripheral membrane protein involved in mitochondrial respiration.

Purpose of the Study:

  • To develop and validate a novel cross-linking method for analyzing phospholipid-protein interactions at the membrane interface.
  • To identify proteins interacting with phospholipids in the inner mitochondrial membrane of Saccharomyces cerevisiae.
  • To apply modern proteomics techniques for capturing these interactions in their native environment.

Main Methods:

  • Utilized a photoactivatable phospholipid analogue (N-(4-azidosalicylamidyl)-1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, ASA-DLPE) for selective cross-linking.

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Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells
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Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

Published on: April 9, 2018

Related Experiment Videos

Last Updated: Jul 16, 2026

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
10:49

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Published on: September 20, 2016

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells
14:02

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

Published on: April 9, 2018

  • Employed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for detection.
  • Applied liquid chromatography tandem mass spectrometry (LC-MS/MS) for protein identification in yeast mitochondrial membranes.

    • Main Results:

    • Successfully cross-linked cytochrome c to ASA-DLPE in a model membrane system, demonstrating method validation.
    • Detected cross-link products of apocytochrome c and endogenous proteins (approx. 34 and 72 kDa) in Saccharomyces cerevisiae inner mitochondrial membranes.
    • Identified candidate proteins potentially interacting at the membrane interface using LC-MS/MS.

    Conclusions:

    • The developed ASA-DLPE cross-linking method effectively captures phospholipid-protein interactions in native biomembranes.
    • This approach provides valuable insights into the molecular organization of the inner mitochondrial membrane.
    • The methodology facilitates the study of membrane-associated protein complexes using advanced proteomics.