Global Mapping of Protein–Lipid Interactions by Using Modified Choline‐Containing Phospholipids Metabolically Synthesized in Live Cells

Abstract

The protein-lipid interaction is an essential metabolic process that mediates cellular signaling and functions. Existing strategies for large-scale mapping studies of the protein-lipid interaction fall short in their incompatibility with metabolic incorporation or inability to remove unwanted interferences from lipidated proteins. By incorporating an alkyne-containing choline head group and a diazirine-modified fatty acid simultaneously into choline-containing phospholipids synthesized from live mammalian cells, proteinphospholipid interactions have been successfully imaged in live cells. Subsequent in situ profiling of the modified Cho phospholipid-crosslinked proteins followed by quantitative proteomics allowed identification of several hundred putative phospholipid-interacting proteins, some of which were further validated.

Greasing the Wheels of Lipid Biology with Chemical Tools

“…The abundance of choline-containing lipids within the cell also made azidoethylcholine an attractive probe of choice for visualizing metabolically labeled nonprotein biomolecules via electron microscopy[25]. Propargylcholine labeling can also be combined with functionalized fatty acid labeling, such as diazirine fatty acids, to allow cellular metabolism to produce multifunctional lipid probes [26].…”

Phosphorus-Based Probes as Molecular Tools for Proteome Studies: Recent Advances in Probe Development and Applications

“…An alternative is based on the utilization of labeled building blocks for their metabolic incorporation into phospholipid structures, as was recently done in studies of protein−phosphatidylcholine interactions. 102 The position of the reporter tag in a phospholipid scaffold has a significant impact on the observed interaction profile. The physicochemical properties of lipids (hydrophobic tail and hydrophilic head) force an appropriate arrangement of the probe in the membrane, which may result in a diverse labeling of proteins on the surface and within the core of the membrane.…”

Comparative Photoaffinity Profiling of Omega-3 Signaling Lipid Probes Reveals Prostaglandin Reductase 1 as a Metabolic Hub in Human Macrophages

“…The ligation handle is used to attach a reporter group via bio-orthogonal chemistry, which allows for visualization or isolation of the probe-bound protein in a complex biological sample. This affinity-based protein profiling (AfBPP) approach has been reported for multiple lipid classes, including phospholipids, 25,26 fatty acids, 23,27 sphingolipids, 28,29 and sterols, 24,30 but has not been applied to omega-3 PUFAs, arguably due to the synthetic challenges associated with their preparation. 31 An important drawback of photoreactive lipid-based probes is their inherent high lipophilicity and nonspecific binding to proteins.…”