In a paper published in Nature 2020 by Patel et al., it was suggested that cholesterol analogs may be useful tools for improving the intracellular delivery of mRNA-encapsulating lipid nanoparticles (LNP) thereby improving the utility of LNP as a delivery vehicle for important RNA-based drugs. In brief, the analogs may improve delivery of the nucleic acids by shifting the movement of the therapeutic molecules into the cytosol rather than into degradation pathways. Aside from this use, cholesterol also plays an important role in membrane biology and as a substrate for many steroids. However, there is a wide range of cholesterol mimetics, with different structural properties, and tools to study which cellular proteins bind to which analogs are key. In a recent article by PEAKS users, researchers use “middle-down” proteomics, the MS-based assessment of medium-sized peptides, to assess different cholesterol mimetics as photolabeling reagents, tools used to evaluate protein-protein interactions in situ. Unlike bottom-up proteomics, which uses proteolytic enzymes to digest proteins into small peptides, middle-down proteomics uses limited digestion to produce longer peptides that, in theory, are more likely to be unique, thus improving sequencing coverage, and leading to improved detection of PTMs and proteoforms. In this study by Krishan et al., researchers demonstrate the use of PEAKS Studio for analyzing middle-down data and identify where the various mimetics bound to their model protein Gloeobacter ligand-gated ion channel (GLIC). This MS method provide 100% sequence coverage of the target protein and allowed the authors to pinpoint the residues where each adduct formed.
Krishnan, Kathiresan, et al. “Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents.” ACS Chemical Biology, American Chemical Society (ACS), Aug. 2021. Crossref, doi:10.1021/acschembio.1c00364. PMID: 34355883.
Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.