Biotinylated probes for the analysis of protein modification by electrophiles.

Codreanu SG, Kim HY, Porter NA, Liebler DC
Methods Mol Biol. 2012 803: 77-95

PMID: 22065220 · PMCID: PMC3811082 · DOI:10.1007/978-1-61779-364-6_7

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. We describe selective adduct capture using biotin affinity probes to enrich protein and peptide adducts for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One approach employs biotinamidohexanoic acid hydrazide to covalently label residual carbonyl groups on adducts. The other employs alkynyl analogs of lipid electrophiles, which 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.

MeSH Terms (21)

Aldehydes Amino Acid Sequence Biochemistry Biotin Biotinylation Blood Proteins Blotting, Western Cell Extracts Cell Line, Tumor Databases, Protein Humans Immunoblotting Indicators and Reagents Mass Spectrometry Molecular Probes Molecular Sequence Data Peptides Protein Processing, Post-Translational Proteins Streptavidin Trypsin

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