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Affinity-labeled electrophile probes

The formation of covalent protein adducts by lipid electrophiles contributes to diseases and toxicities linked to oxidative stress, but analysis of the adducts presents a challenging analytical problem.  Our previous studies employed model biotinyl electrophiles, which enabled capture of adducts on streptavidin.  However, the need to study relevant electrophile chemotypes led us to use analogs without bulky biotin tags, yet which could be biotinylated during sample workup.   Our principal approach employs alkynyl analogs of electrophiles, such as the lipid electrophiles alkynyl-4-hydroxynonenal (aHNE) and alkynyl-4-oxononenal (aONE).  These compounds have toxic potency similar to the alkyl analogs HNE and ONE, but form adducts that can be postlabeled with azidobiotin tags by Cu+-catalyzed cycloaddition (Click chemistry).  To enhance the selectivity of adduct capture we use an azidobiotin reagent with a photocleavable linker, which allows recovery of adducted proteins and peptides under mild conditions.  This approach allows both the identification of protein targets of lipid electrophiles and sequence mapping of the adducts.  These studies have been done with the laboratory of Ned Porter in the Department of Chemistry, who designed and synthesized the alkynyl analogs and biotin probes.  They have been employed by the Liebler laboratory in collaboration with the Porter group and the group of Larry Marnett in the Department of Biochemistry.

Representative references from our work and collaborations

Vila, A., Tallman, K. A., Jacobs, A. T., Liebler, D. C., Porter, N. A., and Marnett, L. J. (2008) Identification of protein targets of 4-hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives. Chem. Res. Toxicol., 21, 432-444.  PubMed

Kim, H. Y., Tallman, K. A., Liebler, D. C., and Porter, N. A. (2009) An azido-biotin reagent for use in the isolation of protein adducts of lipid-derived electrophiles by streptavidin catch and photorelease. Mol. Cell. Proteomics, 8, 2080-2089.  PubMed