Photocrosslinking and Click Chemistry Enable the Specific Detection of Proteins Interacting with Phospholipids at the Membrane Interface

Abstract

New lipid analogs mimicking the abundant membrane phospholipid phosphatidylcholine were developed to photocrosslink proteins interacting with phospholipid headgroups at the membrane interface. In addition to either a phenylazide or benzophenone photoactivatable moiety attached to the headgroup, the lipid analogs contained azides attached as baits to the acyl chains. After photocrosslinking in situ in the biomembrane, these baits were used for the attachment of a fluorescent tetramethylrhodamine-alkyne conjugate or a biotin-alkyne conjugate using click chemistry, allowing for the selective detection and purification of crosslink products, respectively. Proteins crosslinked to the lipid analogs in inner mitochondrial membranes from Saccharomyces cerevisiae were detected and subsequently identified by mass spectrometry. Established interaction partners of phosphatidylcholine were found, as well as known integral and peripheral inner membrane proteins, and proteins that were not previously considered mitochondrial inner membrane proteins.

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

“…Compared to anionic ASA-DLPE, 59 the zwitterionic PC probes were much less effective in crosslinking the positively charged protein cytochrome c. The bifunctional PC analogues were validated by demonstrating that membrane-bound apo-cytochrome c, the unfolded, more hydrophobic precursor of cytochrome c, was rendered carbonate wash resistant after irradiation. 60 Fig . 3 shows the sequence of steps in a crosslink experiment followed by fluorescence detection or purification of the crosslink products.…”

“…In this respect, it is of interest to compare the results obtained with the anionic ASA-DLPE probe 59 to the results obtained for the zwitterionic PC analogues. 60 The difference in headgroup charge was reflected by the very limited overlap (Gut2p and Cyt1p) between proteins crosslinked by ASA-PE and by phenylazide-PC, both containing a phenylazide moiety.…”

“…59 A PC analogue in which the TPD group was attached to the head group did not crosslink proteins, most likely because the short-lived carbene-species generated immediately reacted with water. 60 The ASA-PE probe molecule was validated by demonstrating that UV activation rendered the binding of the positively charged peripheral membrane protein cytochrome c to negatively charged liposomes resistant to carbonate wash. This result was recapitulated in mitochondrial inner membrane vesicles (IMV) in which ASA-PE had been incorporated (75 pmol added per mg IMV protein).…”

Overcoming Key Technological Challenges in Using Mass Spectrometry for Mapping Cell Surfaces in Tissues

“…Although the method appears intuitive, there may be problems relating to the lysis of the cells by the proteases, thus contaminating the membrane "peptide" solution with intracellular proteins. An alternative approach is the cross-linking of PM protein complexes where specific reagents are used to maintain protein complexes in their close to native state (18,19). The cells are subsequently lysed, and the non-complexed proteins can be removed by various methods, including size exclusion chromatography.…”

Development and Leading-Edge Application of Innovative Photoaffinity Labeling

“…To buck-up the missing tag, post-labeling approaches were developed by application of click chemistry. [60][61][62][63] A diol-type diazirine 48 was utilized to introduce an aldehyde by periodate oxidation followed by selective coupling with biotinyl hydrazide to detect trace amounts of labeled product. 64) Preinstalled alkylazide tag on 49 was substantially stable to the photolytic 65) The postlabeling approaches require, however, large excess reagents to complete enough tag on the labeled proteins existing very low concentrations.…”