Skip to main content

Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine

Abstract

Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication of efforts. A major limitation associated with this methodology includes problematic matching up of gels and blots due to the differences in processing and/or image acquisition. In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions.

Abbreviations

FHA:

fluorescent hydroxylamine

IEF:

isoelectric focusing

MALDI:

Matrix-assisted laser desorption/ionization

References

  1. Harman D. Aging: a theory based on free radical and radiation chemsitry. J. Gerontol. 1956;11:298–300.

    PubMed  CAS  Google Scholar 

  2. Poon HF, Calabrese V, Scapagnini G, Butterfield DA. Free radicals: key to brain aging and heme oxygenase as a cellular response to oxidative stress. J Gerontol A Biol Sci Med Sci 2004;59(5): 478–93.

    PubMed  Google Scholar 

  3. Poon HF, Calabrese V, Scapagnini G, Butterfield DA. Free radicals and brain aging. Clin Geriatr Med 2004;20(2):329–59.

    Article  PubMed  Google Scholar 

  4. Stadtman ER. Protein oxidation and aging. Science 1992;257(5074):1220–4.

    Article  PubMed  CAS  Google Scholar 

  5. Keller JN, Huang FF, Zhu H, Yu J, Ho YS, Kindy TS. Oxidative stress-associated impairment of proteasome activity during ischemia-reperfusion injury. J Cereb Blood Flow Metab 2000;20(10): 1467–73.

    Article  PubMed  CAS  Google Scholar 

  6. Poon HF, Vaishnav RA, Getchell TV, Getchell ML, Butterfield DA. Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice. Neurobiol Aging 2006;27(7):1010–9.

    Article  PubMed  CAS  Google Scholar 

  7. Dalle-Donne I, Aldini G, Carini M, Colombo R, Rossi R, Milzani A. Protein carbonylation, cellular dysfunction, and disease progression. J Cell Mol Med 2006;10(2):389–406.

    Article  PubMed  CAS  Google Scholar 

  8. Jones DP. Redefining oxidative stress. Antioxid Redox Signal 2006;8(9–10):1865–79.

    Article  PubMed  CAS  Google Scholar 

  9. Shacter E, Williams JA, Lim M, Levine RL. Differential susceptibility of plasma proteins to oxidative modification: examination by western blot immunoassay. Free Radic Biol Med 1994;17(5):429–37.

    Article  PubMed  CAS  Google Scholar 

  10. Butterfeld DA, Sultana R, Poon HF. Redox Proteomics: A New Approach to Investigate Oxidative Stress in Alzheimer’s Disease. In: Luo Y, Parker L, editors. Neurodegenerative Disorders, Aging and Antioxidants. New York: Marcel Dekker Inc.; 2006.

    Google Scholar 

  11. Efrat A, Hoffmann F, Kriegel K, Schultz C, Wenk C. Geometric algorithms for the analysis of 2Delectrophoresis gels. J Comput Biol 2002;9(2): 299–315.

    Article  PubMed  CAS  Google Scholar 

  12. Conrad CC, Choi J, Malakowsky CA, Talent JM, Dai R, Marshall P, et al. Identification of protein carbonyls after two-dimensional electrophoresis. Proteomics 2001;1(7):829–34.

    Article  PubMed  CAS  Google Scholar 

  13. Conrad CC, Talent JM, Malakowsky CA, Gracy RW. Post-Electrophoretic Identification of Oxidized Proteins. Biol Proced Online 2000;2:39–45.

    Article  PubMed  Google Scholar 

  14. Poon HF, Farr SA, Banks WA, Pierce WM, Klein JB, Morley JE, et al. Proteomic identification of less oxidized brain proteins in aged senescence-accelerated mice following administration of antisense oligonucleotide directed at the Abeta region of amyloid precursor protein. Brain Res Mol Brain Res 2005;138(1):8–16.

    Article  PubMed  CAS  Google Scholar 

  15. Poon HF, Joshi G, Sultana R, Farr SA, Banks WA, Morley JE, et al. Antisense directed at the Abeta region of APP decreases brain oxidative markers in aged senescence accelerated mice. Brain Res 2004;1018(1):86–96.

    Article  PubMed  CAS  Google Scholar 

  16. Poon HF, Castegna A, Farr SA, Thongboonkerd V, Lynn BC, Banks WA, et al. Quantitative proteomics analysis of specific protein expression and oxidative modification in aged senescence-accelerated-prone 8 mice brain. Neuroscience 2004;126(4):915–26.

    Article  PubMed  CAS  Google Scholar 

  17. Clauser KR, Baker P, Burlingame AL. Role of accurate mass measurement (+/− 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal Chem 1999;71(14):2871–82.

    Article  PubMed  CAS  Google Scholar 

  18. Climent I, Tsai L, Levine RL. Derivatization of gamma-glutamyl semialdehyde residues in oxidized proteins by fluoresceinamine. Anal Biochem 1989;182(2):226–32.

    Article  PubMed  CAS  Google Scholar 

  19. Atamna H, Cheung I, Ames BN. A method for detecting abasic sites in living cells: age-dependent changes in base excision repair. Proc Natl Acad Sci U S A 2000;97(2):686–91.

    Article  PubMed  CAS  Google Scholar 

  20. Ide H, Akamatsu K, Kimura Y, Michiue K, Makino K, Asaeda A, et al. Synthesis and damage specificity of a novel probe for the detection of abasic sites in DNA. Biochemistry 1993;32(32): 8276–83.

    Article  PubMed  CAS  Google Scholar 

  21. Kubo K, Ide H, Wallace SS, Kow YW. A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion. Biochemistry 1992;31(14):3703–8.

    Article  PubMed  CAS  Google Scholar 

  22. Chavez J, Wu J, Han B, Chung WG, Maier CS. New role for an old probe: affinity labeling of oxylipid protein conjugates by N′-aminooxymethylcarbonylhydrazino d-biotin. Anal Chem 2006;78(19):6847–54.

    Article  PubMed  CAS  Google Scholar 

  23. DeFilippi LJ, Toler LS, Hultquist DE. Reactivities of hydroxylamine and sodium bisulphite with carbonyl-containing haems and with the prosthetic groups of the erythrocyte green haemoproteins. Biochem J 1979;179(1):151–60.

    PubMed  CAS  Google Scholar 

  24. Poon HF, Hensley K, Thongboonkerd V, Merchant ML, Lynn BC, Pierce WM, et al. Redox proteomics analysis of oxidatively modified proteins in G93A-SOD1 transgenic mice—a model of familial amyotrophic lateral sclerosis. Free Radic Biol Med 2005;39(4):453–62.

    Article  PubMed  CAS  Google Scholar 

  25. Choi J, Malakowsky CA, Talent JM, Conrad CC, Gracy RW. Identification of oxidized plasma proteins in Alzheimer’s disease. Biochem Biophys Res Commun 2002;293(5):1566–70.

    Article  PubMed  CAS  Google Scholar 

  26. Tezel G, Yang X, Cai J. Proteomic identification of oxidatively modified retinal proteins in a chronic pressure-induced rat model of glaucoma. Invest Ophthalmol Vis Sci 2005;46(9):3177–87.

    Article  PubMed  Google Scholar 

  27. Sagt CM, Muller WH, van der Heide L, Boonstra J, Verkleij AJ, Verrips CT. Impaired cutinase secretion in Saccharomyces cerevisiae induces irregular endoplasmic reticulum (ER) membrane proliferation, oxidative stress, and ER-associated degradation. Appl Environ Microbiol 2002;68(5): 2155–60.

    Article  PubMed  CAS  Google Scholar 

  28. Poon HF, Frasier M, Shreve N, Calabrese V, Wolozin B, Butterfield DA. Mitochondrial associated metabolic proteins are selectively oxidized in A30P alpha-synuclein transgenic mice-a model of familial Parkinson’s disease. Neurobiol Dis 2005;18(3):492–8.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to H. Fai Poon.

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Poon, H.F., Abdullah, L., Reed, J. et al. Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine. Biol. Proced. Online 9, 65–72 (2007). https://doi.org/10.1251/bpo134

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo134

Indexing terms