Abstract
HNE (4-hydroxynonenal), a byproduct of lipid peroxidation, reacts with nucleophilic centers on proteins. A terminal alkynyl analog of HNE (alkynyl HNE, aHNE) serves as a surrogate for HNE itself, both compounds reacting with protein amine and thiol functional groups by similar chemistry. Proteins modified with aHNE undergo reaction with a click reagent that bears azido and biotin groups separated by a photocleavable linker. Peptides and proteins modified in this way are affinity purified on streptavidin beads. Photolysis of the beads with a low intensity UV light releases bound biotinylated proteins or peptides, i.e. proteins or peptides modified by aHNE. Two strategies, (a) protein catch and photorelease and (b) peptide catch and photorelease, are employed to enrich adducted proteins or peptide mixtures highly enriched in adducts. Proteomics analysis of the streptavidin-purified peptides by LC-MS/MS permits identification of the adduction site. Identification of 30 separate peptides from human serum albumin by peptide catch and photorelease reveals 18 different aHNE adduction sites on the protein. Protein catch and photorelease shows that both HSA and ApoA1 in human plasma undergo significant modification by aHNE.
Highlights
HNE (4-hydroxynonenal), a byproduct of lipid peroxidation, reacts with nucleophilic centers on proteins
We recently reported that 4-hydroxynon-2-en-8-ynal, alkynyl-HNE, can be used as an HNE surrogate in whole cells to isolate proteins that are adducted by this electrophile [19]. aHNE displays similar toxicity in RKO cells as does HNE, and studies with model peptides and isolated proteins show that HNE and the alkynyl surrogate display similar chemistry in reactions with protein nucleophiles
Reaction of cellular aHNE protein adducts with an azidobiotin reagent followed by capture of the triazole cycloadducts on streptavidin beads permitted a number of adducted proteins to be identified by shotgun proteomics [19]
Summary
Materials—Streptavidin and Alexa Fluorா680-conjugated fluorescent secondary antibodies were obtained from Molecular Probes (Eugene, OR). Protein Catch and Photorelease of HSA Adducts—A filtered reaction mixture was immobilized on a streptavidin slurry (500 l) overnight in the cold (4 °C) in the dark. After filtration using Amicon filter (MWCO ϭ 10 kDa), the proteins were immobilized on a streptavidin slurry (500 l) overnight in the cold room in the dark. The eluted proteins were resolved by SDS-PGE on a 10% BisTris gel, and one was stained with Blueா (Invitrogen), and the other was transferred to a polyvinylidene difluoride membrane for Western blotting to confirm that the click cycloaddition had occurred using Alexa Fluor 680 streptavidin. The reaction mixture was filtered with Amicon (10 kDa) filter, and the recovered proteins were reduced, alkylated, and digested in solution with trypsin as described above. Parsimony rules were applied to generate a minimal list of proteins that explained all of the peptides that passed our entry criteria
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