Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal

Abstract

Lipid peroxidation yields a variety of electrophiles, which are thought to contribute to the molecular pathogenesis of diseases involving oxidative stress, yet little is known of the scope of protein damage caused by lipid electrophiles. We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 mum HNE. HNE Michael adducts were biotinylated by reaction with biotinamidohexanoic acid hydrazide, captured with streptavidin, and the captured proteins were resolved by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and identified by liquid chromatography-tandem mass spectrometry. Of the 1500+ proteins identified, 417 displayed a statistically significant increase in adduction with increasing HNE exposure concentration. We further identified 18 biotin hydrazide-modified, HNE-adducted peptides by specific capture using anti-biotin antibody and analysis by high resolution liquid chromatography-tandem mass spectrometry. A subset of the identified HNE targets were validated with a streptavidin capture and immunoblotting approach, which enabled detection of adducts at HNE exposures as low as 1 mum. Protein interaction network analysis indicated several subsystems impacted by endogenous electrophiles in oxidative stress, including the 26 S proteasomal and chaperonin containing TCP-1 (CCT) systems involved in protein-folding and degradation, as well as the COP9 signalosome, translation initiation complex, and a large network of ribonucleoproteins. Global analyses of protein lipid electrophile adducts provide a systems-level perspective on the mechanisms of diseases involving oxidative stress.

Fig. 1.

A, biotinylation of protein reactive carbonyls with biotin hydrazide. B, experimental flow chart for identification of protein targets of HNE in RKO cells.

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.

Fig. 3.

Protein communities identified from the network of HNE-adducted proteins.

Fig. 4.

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 (arrows), which contained adducted proteins.

Fig. 5.

Semiquantitative analysis of three protein targets, GSTP, actin, and TrxRd1, based on spectral counts (blue) and immunoblot band intensity (purple). Spectral count plots depict mean ± S. D.

Fig. 6.

Detection of biotinylated proteins from RKO cells treated with 0, 1, 5, 10, and 50 μm HNE and then derivatized with biotin hydrazide. A, streptavidin immunoblot of total RKO cell protein extract with increasing protein labeling with HNE exposure concentration. B, immunoblot detection of the indicated proteins following RKO cell treatment with HNE, biotin hydrazide derivatization, capture of biotinylated proteins on streptavidin beads, and elution as described under “Materials and Methods.”