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

Codreanu SG, Zhang B, Sobecki SM, Billheimer DD, Liebler DC - Mol. Cell Proteomics (2008)

Bottom Line: We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 mum HNE.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

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.

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Related in: MedlinePlus

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.
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f5: 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.

Mentions: The identities of some of the protein targets discovered by the LC-MS-MS analyses were validated by targeted measurements of the protein adducts using a combined biotin-avidin capture and immunoblotting approach. RKO cells were treated with HNE and then the adducted proteins were derivatized by reaction with biotin hydrazide and captured with streptavidin as described above. Both the flow-through and elution fractions were collected and subjected to immunoblot analysis with antibodies directed against proteins that had been detected by LC-MS-MS as putative HNE targets. Biotinylated proteins were eluted from the streptavidin beads by boiling them in LDS loading buffer. Blots were probed for HSP90, actin, tubulin, cofilin, GSTP, GAPDH, TrxRd1, Prdx6, cullin3, and bax. Treatment of RKO cells with 0, 50, 100 μm HNE was used to generate RKO protein adducts as for the LC-MS-MS-based studies described above. Examination of these blots clearly shows differences in protein adduction between 0, 50, and 100 μm HNE treatment (Fig. 4). Proteins adducted by HNE were detected by immunoblotting in the elution (E) fractions (Fig. 4, denoted by arrows), which contained adducted proteins. Immunoblot band intensities for the eluted fractions increased with HNE concentrations, and the changes in immunoblot intensity matched changes in adduction observed by spectral counting in LC-MS-MS (Fig. 5). We note that although this approach clearly indicates protein adduction changes, it may be possible to improve the quantitative precision of the assay by incorporating a fixed amount of some biotinylated protein (e.g. albumin) as an internal standard.


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

Codreanu SG, Zhang B, Sobecki SM, Billheimer DD, Liebler DC - Mol. Cell Proteomics (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667350&req=5

f5: 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.
Mentions: The identities of some of the protein targets discovered by the LC-MS-MS analyses were validated by targeted measurements of the protein adducts using a combined biotin-avidin capture and immunoblotting approach. RKO cells were treated with HNE and then the adducted proteins were derivatized by reaction with biotin hydrazide and captured with streptavidin as described above. Both the flow-through and elution fractions were collected and subjected to immunoblot analysis with antibodies directed against proteins that had been detected by LC-MS-MS as putative HNE targets. Biotinylated proteins were eluted from the streptavidin beads by boiling them in LDS loading buffer. Blots were probed for HSP90, actin, tubulin, cofilin, GSTP, GAPDH, TrxRd1, Prdx6, cullin3, and bax. Treatment of RKO cells with 0, 50, 100 μm HNE was used to generate RKO protein adducts as for the LC-MS-MS-based studies described above. Examination of these blots clearly shows differences in protein adduction between 0, 50, and 100 μm HNE treatment (Fig. 4). Proteins adducted by HNE were detected by immunoblotting in the elution (E) fractions (Fig. 4, denoted by arrows), which contained adducted proteins. Immunoblot band intensities for the eluted fractions increased with HNE concentrations, and the changes in immunoblot intensity matched changes in adduction observed by spectral counting in LC-MS-MS (Fig. 5). We note that although this approach clearly indicates protein adduction changes, it may be possible to improve the quantitative precision of the assay by incorporating a fixed amount of some biotinylated protein (e.g. albumin) as an internal standard.

Bottom Line: We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 mum HNE.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

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.

Show MeSH
Related in: MedlinePlus