Identification of Protein Targets of 4-Hydroxynonenal Using Click Chemistry for ex Vivo Biotinylation of Azido and Alkynyl Derivatives

Abstract

Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress. Diffusible electrophilic R, -unsaturated aldehydes, such as 4-hydroxynonenal (HNE), have been shown to modify proteins that mediate cell signaling (e.g., IKK and Keap1) and alter gene expression pathways responsible for inducing antioxidant genes, heat shock proteins, and the DNA damage response. To fully understand cellular responses to HNE, it is important to determine its protein targets in an unbiased fashion. This requires a strategy for detecting and isolating HNE-modified proteins regardless of the nature of the chemical linkage between HNE and its targets. Azido or alkynyl derivatives of HNE were synthesized and demonstrated to be equivalent to HNE in their ability to induce heme oxygenase induction and induce apoptosis in colon cancer (RKO) cells. Cells exposed to the tagged HNE derivatives were lysed and exposed to reagents to effect Staudinger ligation or copper-catalyzed Huisgen 1,3 dipolar cycloaddition reaction (click chemistry) to conjugate HNE-adducted proteins with biotin for subsequent affinity purification. Both strategies yielded efficient biotinylation of tagged HNE-protein conjugates, but click chemistry was found to be superior for the recovery of biotinylated proteins from streptavidincoated beads. Biotinylated proteins were detected in lysates from RKO cell incubations with azido-HNE at concentrations as low as 1 µM. These proteins were affinity purified with streptavidin beads, and proteomic analysis was performed by linear ion trap mass spectrometry. Proteomic analysis revealed a dose-dependent increase in labeled proteins with increased sequence coverage at higher concentrations. Several proteins involved in stress signaling (heat shock proteins 70 and 90 and the 78-kDa glucoseregulated protein) were selectively adducted by azido-and alkynyl-HNE. The use of azido and alkynyl derivatives in conjunction with click chemistry appears to be a valuable approach for the identification of the protein targets of HNE.

Global Analysis of Protein Damage by the Lipid Electrophile 4-Hydroxy-2-nonenal

“…Our preliminary experiments and other recent work (51) indicated that shotgun proteomic analysis of RKO cells exposed to lower HNE concentrations yielded far fewer protein target identifications. Use of higher HNE concentrations enabled detection of greater numbers of adducts, but the relative distributions of adducts should be largely independent of HNE concentration in the concentration range of our experiments.…”

“…Selective analysis of protein adducts requires an affinity capture method. Here we employed biotin hydrazide to label HNE adducts, but other approaches employing affinity postlabeling via Click chemistry or similar methods are certainly applicable (28,51,56). Although affinity capture enriches for adducted proteins, nonspecific binding of proteins to affinity-capture medium results in false positive target identifications.…”

Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds

“…For example, Vila et al worked with alkynyl analogues of HNE to form adducts that can be label with azidobiotin tags by Cu + -catalyzed cycloaddition (Click chemistry), and they found that heat shock proteins 70 and 90, and the 78-kDa glucose-regulated protein, were selectively adducted [127]. While clearly a very effective approach to determining targets of HNE, unfortunately it cannot as yet be used to study the physiological occurrence of aldehydes in biological or clinical samples.…”

Redox Proteomics in Selected Neurodegenerative Disorders: From Its Infancy to Future Applications

“…Enrichment of HNE-modified proteins. Several of the approaches for the chemical enrichment of PCO-modified proteins can be applied for the identification of HNE modifications, such as biotin functionalized probes (98,275,392). For example, the biotin functionalized probe N¢aminoox-ymethylcarbonylhydrazino-D-biotin, also known as aldehydereactive probe, has been applied to identify HNE-modified proteins in human monocyte cell lines before and after ascorbic acid treatment (97).…”