Skip to main content
SpringerLink
Log in

How to Use 2D Gel Electrophoresis in Plant Proteomics

  • Protocol
  • First Online:
Plant Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1072))

Abstract

Two-dimensional electrophoresis has nurtured the birth of proteomics. It is however no longer the exclusive setup used in proteomics, with the development of shotgun proteomics techniques that appear more fancy and fashionable nowadays.

Nevertheless, 2D gel-based proteomics still has valuable features, and sometimes unique ones, which make it often an attractive choice when a proteomics strategy must be selected. These features are detailed in this chapter, as is the rationale for selecting or not 2D gel-based proteomics as a proteomic strategy.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Santoni V, Molloy M, Rabilloud T (2000) Membrane proteins and proteomics: un amour impossible? Electrophoresis 21:1054–1070

    Article  PubMed  CAS  Google Scholar 

  2. Rabilloud T (2009) Membrane proteins and proteomics: love is possible, but so difficult. Electrophoresis 30(Suppl 1):S174–S180

    Article  PubMed  Google Scholar 

  3. Santoni V, Rabilloud T, Doumas P et al (1999) Towards the recovery of hydrophobic proteins on two-dimensional electrophoresis gels. Electrophoresis 20:705–711

    Article  PubMed  CAS  Google Scholar 

  4. Santoni V, Kieffer S, Desclaux D et al (2000) Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. Electrophoresis 21:3329–3344

    Article  PubMed  CAS  Google Scholar 

  5. Petrak J, Ivanek R, Toman O et al (2008) Deja vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins. Proteomics 8:1744–1749

    Article  PubMed  CAS  Google Scholar 

  6. Wang P, Bouwman FG, Mariman EC (2009) Generally detected proteins in comparative proteomics—a matter of cellular stress response? Proteomics 9:2955–2966

    Article  PubMed  CAS  Google Scholar 

  7. Nesvizhskii AI, Aebersold R (2005) Interpretation of shotgun proteomic data: the protein inference problem. Mol Cell Proteomics 4:1419–1440

    Article  PubMed  CAS  Google Scholar 

  8. Li YF, Arnold RJ, Li Y et al (2009) A bayesian approach to protein inference problem in shotgun proteomics. J Comput Biol 16:1183–1193

    Article  PubMed  CAS  Google Scholar 

  9. Grobei MA, Qeli E, Brunner E et al (2009) Deterministic protein inference for shotgun proteomics data provides new insights into Arabidopsis pollen development and function. Genome Res 19:1786–1800

    Article  PubMed  CAS  Google Scholar 

  10. Choe LH, Lee KH (2003) Quantitative and qualitative measure of intralaboratory two-dimensional protein gel reproducibility and the effects of sample preparation, sample load, and image analysis. Electrophoresis 24:3500–3507

    Article  PubMed  CAS  Google Scholar 

  11. Zhou SB, Bailey MJ, Dunn MJ et al (2005) A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure. Proteomics 5:2739–2747

    Article  PubMed  CAS  Google Scholar 

  12. Saravanan RS, Rose JK (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4:2522–2532

    Article  PubMed  CAS  Google Scholar 

  13. Sarry JE, Kuhn L, Ducruix C et al (2006) The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses. Proteomics 6:2180–2198

    Article  PubMed  CAS  Google Scholar 

  14. Villiers F, Ducruix C, Hugouvieux V et al (2011) Investigating the plant response to cadmium exposure by proteomic and metabolomic approaches. Proteomics 11:1650–1663

    Article  PubMed  CAS  Google Scholar 

  15. Tanou G, Job C, Belghazi M et al (2011) Proteomic signatures uncover hydrogen peroxide and nitric oxide cross-talk signaling network in citrus plants. J Proteome Res 9:5994–6006

    Article  Google Scholar 

  16. Catusse J, Meinhard J, Job C et al (2011) Proteomics reveals potential biomarkers of seed vigor in sugarbeet. Proteomics 11:1569–1580

    Article  PubMed  CAS  Google Scholar 

  17. Hackett M (2008) Science, marketing and wishful thinking in quantitative proteomics. Proteomics 8:4618–4623

    Article  PubMed  CAS  Google Scholar 

  18. Celis JE (2004) Gel-based proteomics: what does MCP expect? Mol Cell Proteomics 3:949

    PubMed  CAS  Google Scholar 

  19. Wilkins MR, Appel RD, Van Eyk JE et al (2006) Guidelines for the next 10 years of proteomics. Proteomics 6:4–8

    Article  PubMed  CAS  Google Scholar 

  20. Corbett JM, Dunn MJ, Posch A et al (1994) Positional reproducibility of protein spots in 2-dimensional polyacrylamide-gel electrophoresis using immobilized ph gradient isoelectric-focusing in the first dimension—an interlaboratory comparison. Electrophoresis 15:1205–1211

    Article  PubMed  CAS  Google Scholar 

  21. Blomberg A, Blomberg L, Norbeck J et al (1995) Interlaboratory reproducibility of yeast protein-patterns analyzed by immobilized Ph gradient 2-dimensional gel-electrophoresis. Electrophoresis 16:1935–1945

    Article  PubMed  CAS  Google Scholar 

  22. Paulovich AG, Billheimer D, Ham AJ et al (2010) Interlaboratory study characterizing a yeast performance standard for benchmarking LC-MS platform performance. Mol Cell Proteomics 9:242–254

    Article  PubMed  CAS  Google Scholar 

  23. Tabb DL, Vega-Montoto L, Rudnick PA et al (2010) Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry. J Proteome Res 9:761–776

    Article  PubMed  CAS  Google Scholar 

  24. Anderson NG, Anderson NL (1978) Analytical techniques for cell fractions. 21. 2-Dimensional analysis of serum and tissue proteins—multiple isoelectric-focusing. Anal Biochem 85:331–340

    Article  PubMed  CAS  Google Scholar 

  25. Anderson NL, Anderson NG (1978) Analytical techniques for cell fractions .22. 2-Dimensional analysis of serum and tissue proteins—multiple gradient-slab gel-electrophoresis. Anal Biochem 85:341–354

    Article  PubMed  CAS  Google Scholar 

  26. Gorg A, Postel W, Gunther S (1988) Methodology of Ipg-Dalt for the analysis of cell lysates and tissue proteins. Electrophoresis 9:628

    Google Scholar 

  27. Natera SHA, Guerreiro N, Djordjevic MA (2000) Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis. Mol Plant Microbe Interact 13:995–1009

    Article  PubMed  CAS  Google Scholar 

  28. Miller I, Crawford J, Gianazza E (2006) Protein stains for proteornic applications: which, when, why? Proteomics 6:5385–5408

    Article  PubMed  CAS  Google Scholar 

  29. Patton WF (2000) A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis 21:1123–1144

    Article  PubMed  CAS  Google Scholar 

  30. Minden JS, Dowd SR, Meyer HE et al (2009) Difference gel electrophoresis. Electrophoresis 30:S156–S161

    Article  PubMed  Google Scholar 

  31. Bandow JE, Baker JD, Berth M et al (2008) Improved image analysis workflow for 2-D gels enables large-scale 2-D gel-based proteomics studies—COPD biomarker discovery study. Proteomics 8:3030–3041

    Article  PubMed  CAS  Google Scholar 

  32. Stessl M, Noe CR, Lachmann B (2009) Influence of image-analysis software on quantitation of two-dimensional gel electrophoresis data. Electrophoresis 30:325–328

    Article  PubMed  CAS  Google Scholar 

  33. Karp NA, McCormick PS, Russell MR et al (2007) Experimental and statistical considerations to avoid false conclusions in proteomics studies using differential in-gel electrophoresis. Mol Cell Proteomics 6:1354–1364

    Article  PubMed  CAS  Google Scholar 

  34. Thingholm TE, Jensen ON, Larsen MR (2009) Analytical strategies for phosphoproteomics. Proteomics 9:1451–1468

    Article  PubMed  CAS  Google Scholar 

  35. Robotti A, Natale M, Albo AG et al (2010) Acute-phase proteins investigation based on lectins affinity capture prior to 2-DE separation: application to serum from multiple sclerosis patients. Electrophoresis 31:2882–2893

    Article  PubMed  CAS  Google Scholar 

  36. Ligat L, Lauber E, Albenne C et al (2011) Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins. Proteomics 11:1798–1813

    Article  PubMed  CAS  Google Scholar 

  37. Catala C, Howe KJ, Hucko S et al (2011) Towards characterization of the glycoproteome of tomato (Solanum lycopersicum) fruit using Concanavalin A lectin affinity chromatography and LC-MALDI-MS/MS analysis. Proteomics 11:1530–1544

    Article  PubMed  CAS  Google Scholar 

  38. Zhang Y, Giboulot A, Zivy M et al (2011) Combining various strategies to increase the coverage of the plant cell wall glycoproteome. Phytochemistry 72:1109–1123

    Article  PubMed  CAS  Google Scholar 

  39. Komatsu S, Yamada E, Furukawa K (2009) Cold stress changes the concanavalin A-positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths. Amino Acids 36:115–123

    Article  PubMed  CAS  Google Scholar 

  40. Ishigami A, Ohsawa T, Hiratsuka M et al (2005) Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer’s disease. J Neurosci Res 80:120–128

    Article  PubMed  CAS  Google Scholar 

  41. Job C, Rajjou L, Lovigny Y et al (2005) Patterns of protein oxidation in Arabidopsis seeds and during germination. Plant Physiol 138:790–802

    Article  PubMed  CAS  Google Scholar 

  42. Castegna A, Thongboonkerd V, Klein JB et al (2003) Proteomic identification of nitrated proteins in Alzheimer’s disease brain. J Neurochem 85:1394–1401

    Article  PubMed  CAS  Google Scholar 

  43. Kanski J, Behring A, Pelling J et al (2005) Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging. Am J Physiol Heart Circ Physiol 288:H371–H381

    Article  PubMed  CAS  Google Scholar 

  44. Perluigi M, Fai PH, Hensley K et al (2005) Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A–SOD1 transgenic mice—a model of familial amyotrophic lateral sclerosis. Free Radic Biol Med 38:960–968

    Article  PubMed  CAS  Google Scholar 

  45. Reed TT, Pierce WM, Markesbery WR et al (2009) Proteomic identification of HNE-bound proteins in early Alzheimer disease: insights into the role of lipid peroxidation in the progression of AD. Brain Res 1274:66–76

    Article  PubMed  CAS  Google Scholar 

  46. Parker KC, Garrels JI, Hines W et al (1998) Identification of yeast proteins from two-dimensional gels: working out spot cross-contamination. Electrophoresis 19:1920–1932

    Article  PubMed  CAS  Google Scholar 

  47. Westbrook JA, Yan JX, Wait R et al (2001) Zooming-in on the proteome: very narrow-range immobilised pH gradients reveal more protein species and isoforms. Electrophoresis 22:2865–2871

    Article  PubMed  CAS  Google Scholar 

  48. Sarma AD, Emerich DW (2006) A comparative proteomic evaluation of culture grown vs nodule isolated Bradyrhizobium japonicum. Proteomics 6:3008–3028

    Article  PubMed  CAS  Google Scholar 

  49. Zhao Z, Zhang W, Stanley BA et al (2008) Functional proteomics of Arabidopsis thaliana guard cells uncovers new stomatal signaling pathways. Plant Cell 20:3210–3226

    Article  PubMed  CAS  Google Scholar 

  50. Righetti PG, Castagna A, Herbert B et al (2005) How to bring the “unseen” proteome to the limelight via electrophoretic pre-fractionation techniques. Biosci Rep 25:3–17

    Article  PubMed  CAS  Google Scholar 

  51. Rajjou L, Belghazi M, Catusse J et al (2011) Proteomics and posttranslational proteomics of seed dormancy and germination. Methods Mol Biol 773:215–236

    Article  PubMed  CAS  Google Scholar 

  52. Arc E, Galland M, Cueff G et al (2011) Reboot the system thanks to protein post-translational modifications and proteome diversity: how quiescent seeds restart their metabolism to prepare seedling establishment. Proteomics 11:1606–1618

    Article  PubMed  CAS  Google Scholar 

  53. Santoni V, Verdoucq L, Sommerer N et al (2006) Methylation of aquaporins in plant plasma membrane. Biochem J 400:189–197

    Article  PubMed  CAS  Google Scholar 

  54. Fratelli M, Demol H, Puype M et al (2002) Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc Natl Acad Sci USA 99:3505–3510

    Article  PubMed  CAS  Google Scholar 

  55. Lee AY, Park BC, Jang M et al (2004) Identification of caspase-3 degradome by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics 4:3429–3436

    Article  PubMed  CAS  Google Scholar 

  56. Jang M, Park BC, Kang S et al (2008) Mining of caspase-7 substrates using a degradomic approach. Mol Cells 26:152–157

    PubMed  CAS  Google Scholar 

  57. Rabilloud T, Heller M, Gasnier F et al (2002) Proteomics analysis of cellular response to oxidative stress—evidence for in vivo overoxidation of peroxiredoxins at their active site. J Biol Chem 277:19396–19401

    Article  PubMed  CAS  Google Scholar 

  58. Weber H, Engelmann S, Becher D et al (2004) Oxidative stress triggers thiol oxidation in the glyceraldehyde-3-phosphate dehydrogenase of Staphylococcus aureus. Mol Microbiol 52:133–140

    Article  PubMed  CAS  Google Scholar 

  59. Hwang NR, Yim SH, Kim YM et al (2009) Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions. Biochem J 423:253–264

    Article  PubMed  CAS  Google Scholar 

  60. Hanash SM, Strahler JR, Neel JV et al (1991) Highly resolving 2-dimensional gels for protein sequencing. Proc Natl Acad Sci USA 88:5709–5713

    Article  PubMed  CAS  Google Scholar 

  61. John JP, Pollak A, Lubec G (2009) Complete sequencing and oxidative modification of manganese superoxide in medulloblastoma cells. Electrophoresis 30:3006–3016

    Article  PubMed  CAS  Google Scholar 

  62. Seo J, Jeong J, Kim YM et al (2008) Strategy for comprehensive identification of post-cellular proteins, including low abundant modifications: glyceraldehyde-3-phosphate dehydrogenase. J Proteome Res 7:587–602

    Article  PubMed  CAS  Google Scholar 

  63. Bosisio AB, Rochette J, Wajcman H et al (1985) Electrophoretic and chromatographic techniques for the differential diagnosis of a haemoglobin abnormality: Hb E heterozygosity. J Chromatogr 330:299–306

    Article  PubMed  CAS  Google Scholar 

  64. Cossu G, Righetti PG (1987) Resolution of G gamma and A gamma foetal haemoglobin tetramers in immobilized pH gradients. J Chromatogr 398:211–216

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Chemistry and Biology of Metals, CNRS UMR 5249, Grenoble, France

    Thierry Rabilloud

  2. Université Joseph Fourier, Grenoble, France

    Thierry Rabilloud

  3. iRTSV/LCBM, CEA Grenoble, Grenoble, France

    Thierry Rabilloud

Authors
  1. Thierry Rabilloud
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Dept of Biochem & Molecular Bio, University of Cordoba, Cordoba, Spain

    Jesus V. Jorrin-Novo

  2. University of Tsukuba National Institute of Crop Science, Tsukuba, Japan

    Setsuko Komatsu

  3. Molecular Systems Biology, University of Vienna, Vienna, Austria

    Wolfram Weckwerth

  4. Dept. Molecular Systems Biology, University of Vienna, Vienna, Austria

    Stefanie Wienkoop

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Rabilloud, T. (2014). How to Use 2D Gel Electrophoresis in Plant Proteomics. In: Jorrin-Novo, J., Komatsu, S., Weckwerth, W., Wienkoop, S. (eds) Plant Proteomics. Methods in Molecular Biology, vol 1072. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-631-3_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-631-3_4

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-630-6

  • Online ISBN: 978-1-62703-631-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation