An Azido-Biotin Reagent for Use in the Isolation of Protein Adducts of Lipid-derived Electrophiles by Streptavidin Catch and Photorelease

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. Polyunsaturated lipids in biological membranes are particularly reactive targets for oxygen radicals (1-3). Lipid peroxidation, the chain reaction of peroxyl radicals that is a consequence of oxidative stress, is thought to be involved in human diseases such as cancer, atherosclerosis, and neurodegenerative disorders (4 -8). A variety of electrophilic compounds are byproducts of lipid peroxidation, 4-hydroxynon-2-enal (HNE) 1 being a particularly toxic electrophile (9 -12) that forms mutagenic DNA adducts (13-15). HNE and other lipid-derived electrophiles also form protein modifications, and some of these adducts have been characterized on a limited number of proteins and peptides by mass spectrometry (MS) and in tissues by antibody-based methods (16). Until recently, relatively little was known about the target selectivity of oxidantderived electrophiles in proteins, the relative reactivities of different amino acid targets, and the properties of the adducts. We recently described the application of a post-labeling strategy in which biotin hydrazide was used to biotinylate carbonyl-containing adducts formed by HNE in RKO cells (17). When combined with shotgun proteome analysis of the captured proteins, this approach provided a global perspective on patterns of protein damage by a prototypical lipid electrophile. However, biotin hydrazide labels many carbonyls, thus generating a background inventory derived from endogenous carbonyls, which is difficult to characterize and may mask more subtle patterns of selectivity in protein adduction. Moreover, the biotin hydrazide approach can only capture adducts with a reactive carbonyl group. To deal with these limitations, we have explored labeled electrophile probes and selective adduct capture chemistries (18). We recently reported that 4-hydroxynon-2-en-8-ynal, alkynyl-HNE (aHNE), 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. For example, reaction of aHNE with proteins or peptides followed by sodium borohydride reduction gives Michael and imine adducts as shown in structures 1 and 2. This same chemistry is observed for HNE itself.Reaction ...

Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells

“…All three CFZ analogs exhibited proteasome inhibitory activity comparable to CFZ and compounds OP-829 and OP-830 exhibited similar stability in hepatocytes. Compound OP-829 was chosen for further analysis based on these data and the ready availability of the photocleavable click biotinylation reagent azido-UV-biotin, which we had used previously for similar analyses (30). MS/MS analyses of both CFZ and OP-829 were performed to further confirm their structures and identify the fragmentation patterns of both compounds (supplemental Fig.…”

“…The photocleavable azidobiotin click reagent (azido-UVbiotin) was synthesized as described previously (30).…”

Analysis of protein dynamics at active, stalled, and collapsed replication forks

“…The click reaction buffer contains Invitrogen's Click-iT cell reaction buffer and cell buffer additive (C10269), 2 mM copper (II) sulfate (CuSO 4 ), and 1 mM photocleavable biotinazide (Kim et al 2009) (kindly provided by Ned Porter). DMSO was added instead of biotin-azide to the negative control samples (no clk in all figures).…”

Probing lipid-protein adduction with alkynyl surrogates: application to Smith-Lemli-Opitz syndrome

“…Protein modifi cation with HNE may be involved in the development of various diseases, including Parkinson's disease, Alzheimer's disease, and atherosclerosis, in addition to altered cell signaling (23)(24)(25)(26). HNE-protein adduction has been studied extensively, with protein targets of modifi cation and sites of adduction within modifi ed proteins identifi ed ( 21,27 ).…”