Proteome-wide detection of phospholipid–protein interactions in mitochondria by photocrosslinking and click chemistry

Abstract

Photoactivatable lipid analogues are uniquely suited for the detection of lipid-protein interactions in biological membranes. Based on photocrosslinking, new methodology has been developed for the proteome-wide detection of lipid-protein interactions. Bifunctional lipid analogues containing a tag for click chemistry in addition to the photoactivatable moiety enable the enrichment of the crosslinked proteins that is required for subsequent identification by mass spectrometry. In principle the phospholipid interaction-based membrane protein proteomics approach is applicable to any biomembrane and any lipid. Here, we review the background and the development of the new methodology. Results obtained with photocrosslinking in purified mitochondrial membranes from the yeast Saccharomyces cerevisiae are summarized and future perspectives discussed.

Phosphatidylinositol 3,4,5-Trisphosphate Activity Probes for the Labeling and Proteomic Characterization of Protein Binding Partners

“…Activity probes corresponding to ligands that interact with proteins solely through non-covalent binding interactions typically employ photoaffinity tags 46 to enforce covalent labeling of target proteins. 33,34,47–49 For this purpose, we implemented the benzophenone group due to its robust nature and ease of synthesis. Based on these principles, our designs for activity probes ( 1−3 ) based on PIP n headgroups are shown in Figure 1.…”

Global approaches for the elucidation of phosphoinositide-binding proteins

“…Recently, ABPP has begun to impact lipid studies . Gubbens and co-workers developed a range of PC ABPs containing both a photoaffinity label and click chemistry tags for the proteomic elucidation of PCbinding proteins (Gubbens and de Kroon, 2010;Gubbens et al, 2007Gubbens et al, , 2009). In addition, enzymes that manipulate PC have been labeled using ABPs containing a fluorophosphonate reactive group in place of one acyl chain (Tully and Cravatt, 2010).…”

Turning the spotlight on protein–lipid interactions in cells

“…Among multiple bioorthogonal reactions the most frequently used is copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) [21], as alkyne and azide are synthetically accessible and small enough to minimize possible perturbation to biomolecules in which they are incorporated. The extensive application of bioorthogonal reactions for tracking biomolecules has inspired new bifunctional lipid probes for studying protein-lipid interactions, which, in addition to photoactivatable groups, contain bioorthogonal functionalities such as alkyne or azide as chemical handles [27,32,33]. As illustrated in Figure 1b, following the insertion of a bifunctional lipid probe into biological membranes, UV irradiation activates the photoactivatable group and enables crosslinking of proximate proteins with the probe to form stable protein-lipid complexes.…”

“…The initial demonstration of this strategy for characterization and identification of lipid-binding proteins was described by de Kroon and co-workers in 2009 [32,35••]. To identify proteins interacting with phosphatidylcholine (PC)headgroups at mitochondrial membrane interface, they synthesized bifunctional PC analogs ( 1 , 2 ) containing clickable azides at both acyl chains as well as different photoactivatable groups (benzophenone, phenylazide, or diazirines) on the quaternary ammonium group of PC (Figure 2a).…”