Quantitative Chemoproteomics for Site-Specific Analysis of Protein Alkylation by 4‑Hydroxy-2-Nonenal in Cells

Protein alkylation by 4-hydroxy-2-nonenal
(HNE), an endogenous
lipid derived electrophile, contributes to stress signaling and cellular
toxicity. Although previous work has identified protein targets for
HNE alkylation, the sequence specificity of alkylation and dynamics
in a cellular context remain largely unexplored. We developed a new
quantitative chemoproteomic platform, which uses isotopically tagged,
photocleavable azido-biotin reagents to selectively capture and quantify
the cellular targets labeled by the alkynyl analogue of HNE (aHNE).
Our analyses site-specifically identified and quantified 398 aHNE
protein alkylation events (386 cysteine sites and 12 histidine sites)
in intact cells. This data set expands by at least an order of magnitude
the number of such modification sites previously reported. Although
adducts formed by Michael addition are thought to be largely irreversible,
we found that most aHNE modifications are lost rapidly in
situ. Moreover, aHNE adduct turnover occurs only in intact
cells and loss rates are site-selective. This quantitative chemoproteomics
platform provides a versatile general approach to map bioorthogonal-chemically
engineered post-translational modifications and their cellular dynamics
in a site-specific and unbiased manner.