Biotinylated probes for the analysis of protein modification by electrophiles

Abstract

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.

Fig. 1

(A) Biotinylation of protein reactive carbonyls with biotin hydrazide and (B) biotinylation by click chemistry using a photocleavable probe.

Fig. 2

Overlap of adducted proteins identified from RKO cells treated with 50 or 100 μM HNE or vehicle control. The numbers represent total proteins identified in triplicate analyses of each experimental condition. Overlaps between treatments are indicated by the numbers in the corresponding segments. A total of 561 proteins were common to all groups. (Reproduced from Mol Cell Proteomics. 2009 Apr; 8(4):670-80 with permission from ASBMB.)

Fig. 3

Immunoblotting validation of ten individual protein targets from RKO cellular extracts treated with increasing concentrations of HNE. The presence of the proteins was confirmed in the input (I), flow-through (F) and elution (E) fractions (red arrows), which contain adducted proteins. (Reproduced from Mol Cell Proteomics. 2009 Apr; 8(4):670-80 with permission from ASBMB.)

Fig. 4

Immunoblots of plasma supplemented with aHNE followed by 1, 3 cycloaddtion with azido-biotin then released by photolysis. The final concentrations of reagents, biotin, TCEP (or ascorbate), CuSO₄, and ligand were 1 mM, 2 mM, 2 mM, and 0.2 mM, respectively. (A) anti-HSA visualized with Alexa Fluor 680 donkey anti-goat (B) anti-ApoA1 visualized with Alexa Fluor 680 goat anti-rabbit (C) visualized by Alexa Fluor 680-conjugated with streptavidin: residual biotin is probed even after the photolysis due to multiple sites of adduction. The whole plasma proteins are shown in the crude mix on the left and proteins that are eluted from photolysis of beads on the right. Intensities of + or − aHNE can be compared in each case; photoeluted fractions are diluted compared with crude mix. (Reproduced from Mol Cell Proteomics. 2009 Sep; 8(9):2080-9 with permission from ASBMB.)