Advertisement

Gel-Based and Gel-Free Proteomic Technologies

  • Peter Scherp
  • Ginger Ku
  • Liana Coleman
  • Indu KheterpalEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 702)

Abstract

Proteomics refers to the analysis of expression, localization, functions, posttranslational modifications, and interactions of proteins expressed by a genome at a specific condition and at a specific time. Mass spectrometry (MS)-based proteomic methods have emerged as a key technology for unbiased systematic and high-throughput identification and quantification of complex protein mixtures. These methods have the potential to reveal unknown and novel changes in protein interactions and assemblies that regulate cellular and physiological processes. Both gel-based (one-dimensional [1D] gel electrophoresis, two-dimensional [2D] polyacrylamide gel electrophoresis, 2D difference in-gel electrophoresis [DIGE]) and gel-free (liquid chromatography [LC], capillary electrophoresis) approaches have been developed and utilized in a variety of combinations to separate proteins prior to mass spectrometric analysis. Detailed protocols for global proteomic analysis from adipose-derived stem cells (ASCs) using two central strategies, 2D-DIGE-MS and 2D-LC-MS, are presented here.

Key words

Adipose-derived stem cells Proteomics Mass spectrometry Two-dimensional difference in-gel electrophoresis Liquid chromatography iTRAQ™ 

References

  1. 1.
    Zuk, P. A., Zhu, M., Ashjian, P., De Ugarte, D. A., Huang, J. I., Mizuno, H., Alfonso, Z. C., Fraser, J. K., Benhaim, P., and Hedrick, M. H. (2002) Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13, 4279–95.PubMedCrossRefGoogle Scholar
  2. 2.
    Gimble, J. and Guilak, F. (2003) Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 5, 362–9.PubMedCrossRefGoogle Scholar
  3. 3.
    Bunnell, B. A., Estes, B. T., Guilak, F., and Gimble, J. M. (2008) Differentiation of adipose stem cells. Methods Mol Biol 456, 155–71.PubMedCrossRefGoogle Scholar
  4. 4.
    Celis, J. E., Moreira, J. M., Cabezon, T., Gromov, P., Friis, E., Rank, F., and Gromova, I. (2005) Identification of extracellular and intracellular signaling components of the mammary adipose tissue and its interstitial fluid in high risk breast cancer patients: toward dissecting the molecular circuitry of epithelial-adipocyte stromal cell interactions. Mol Cell Proteomics 4, 492–522.PubMedCrossRefGoogle Scholar
  5. 5.
    DeLany, J. P., Floyd, Z. E., Zvonic, S., Smith, A., Gravois, A., Reiners, E., Wu, X. Y., Kilroy, G., Lefevre, M., and Gimble, J. M. (2005) Proteomic analysis of primary cultures of human adipose-derived stem cells – modulation by adipogenesis. Mol Cell Proteomics 4, 731–40.PubMedCrossRefGoogle Scholar
  6. 6.
    Kim, W. S., Park, B. S., Kim, H. K., Park, J. S., Kim, K. J., Choi, J. S., Chung, S. J., Kim, D. D., and Sung, J. H. (2008) Evidence supporting antioxidant action of adipose-derived stem cells: protection of human dermal fibroblasts from oxidative stress. J Dermatol Sci 49, 133–42.PubMedCrossRefGoogle Scholar
  7. 7.
    Noel, D., Caton, D., Roche, S., Bony, C., Lehmann, S., Casteilla, L., Jorgensen, C., and Cousin, B. (2008) Cell specific differences between human adipose-derived and mesenchymal-stromal cells despite similar differentiation potentials. Exp Cell Res 314, 1575–84.PubMedCrossRefGoogle Scholar
  8. 8.
    Roche, S., Delorme, B., Oostendorp, R. A., Barbet, R., Caton, D., Noel, D., Boumediene, K., Papadaki, H. A., Cousin, B., Crozet, C., Milhavet, O., Casteilla, L., Hatzfeld, J., Jorgensen, C., Charbord, P., and Lehmann, S. (2009) Comparative proteomic analysis of human mesenchymal and embryonic stem cells: towards the definition of a mesenchymal stem cell proteomic signature. Proteomics 9, 223–32.PubMedCrossRefGoogle Scholar
  9. 9.
    Zvonic, S., Lefevre, M., Kilroy, G., Floyd, Z. E., DeLany, J. P., Kheterpal, I., Gravois, A., Dow, R., White, A., Wu, X., and Gimble, J. M. (2007) Secretome of primary cultures of human adipose-derived stem cells: modulation of serpins by adipogenesis. Mol Cell Proteomics 6, 18–28.PubMedGoogle Scholar
  10. 10.
    Cravatt, B. F., Simon, G. M., and Yates, J. R., 3rd (2007) The biological impact of mass-spectrometry-based proteomics. Nature 450, 991–1000.PubMedCrossRefGoogle Scholar
  11. 11.
    Devarajan, P. (2007) Proteomics for biomarker discovery in acute kidney injury. Semin Nephrol 27, 637–51.PubMedCrossRefGoogle Scholar
  12. 12.
    Merchant, M. L. and Klein, J. B. (2007) Proteomics and diabetic nephropathy. Semin Nephrol 27, 627–36.PubMedCrossRefGoogle Scholar
  13. 13.
    Conrotto, P. and Souchelnytskyi, S. (2008) Proteomic approaches in biological and medical sciences: principles and applications. Exp Oncol 30, 171–80.PubMedGoogle Scholar
  14. 14.
    Lambert, J. P., Ethier, M., Smith, J. C., and Figeys, D. (2005) Proteomics: from gel based to gel free. Anal Chem 77, 3771–87.PubMedCrossRefGoogle Scholar
  15. 15.
    Abu-Farha, M., Elisma, F., Zhou, H., Tian, R., Asmer, M. S., and Figeys, D. (2009) Proteomics: from technology developments to biological applications. Anal Chem 81, 4585–99.PubMedCrossRefGoogle Scholar
  16. 16.
    Malmstrom, J., Lee, H., and Aebersold, R. (2007) Advances in proteomic workflows for systems biology. Curr Opin Biotechnol 18, 378–84.PubMedCrossRefGoogle Scholar
  17. 17.
    Ong, S. E. and Mann, M. (2005) Massspectrometry-based proteomics turns quantitative. Nat Chem Biol 1, 252–62.PubMedCrossRefGoogle Scholar
  18. 18.
    Fournier, M. L., Gilmore, J. M., Martin-Brown, S. A., and Washburn, M. P. (2007) Multidimensional separations-based shotgun proteomics. Chem Rev 107, 3654–86.PubMedCrossRefGoogle Scholar
  19. 19.
    O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007–21.PubMedGoogle Scholar
  20. 20.
    Gorg, A., Weiss, W., and Dunn, M. J. (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–85.PubMedCrossRefGoogle Scholar
  21. 21.
    Wittmann-Liebold, B., Graack, H. R., and Pohl, T. (2006) Two-dimensional gel ­electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry. Proteomics 6, 4688–703.PubMedCrossRefGoogle Scholar
  22. 22.
    Marouga, R., David, S., and Hawkins, E. (2005) The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal Bioanal Chem 382, 669–78.PubMedCrossRefGoogle Scholar
  23. 23.
    Kheterpal, I., Coleman, L., Ku, G., Wang, Z. Q., Ribnicky, D., and Cefalu, W. T. (2010) Regulation of insulin action by an extract of Artemisia dracunculus L. in primary human skeletal muscle culture – a proteomics approach. Phytother Res 24, 1278–84.PubMedCrossRefGoogle Scholar
  24. 24.
    Aebersold, R. and Mann, M. (2003) Mass spectrometry-based proteomics. Nature 422, 198–207.PubMedCrossRefGoogle Scholar
  25. 25.
    Gygi, S. P., Rist, B., Griffin, T. J., Eng, J., and Aebersold, R. (2002) Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags. J Proteome Res 1, 47–54.PubMedCrossRefGoogle Scholar
  26. 26.
    Salim, K., Kehoe, L., Minkoff, M. S., Bilsland, J. G., Munoz-Sanjuan, I., and Guest, P. C. (2006) Identification of differentiating neural progenitor cell markers using shotgun isobaric tagging mass spectrometry. Stem Cells Dev 15, 461–70.PubMedCrossRefGoogle Scholar
  27. 27.
    Griffiths, S. D., Burthem, J., Unwin, R. D., Holyoake, T. L., Melo, J. V., Lucas, G. S., and Whetton, A. D. (2007) The use of isobaric tag peptide labeling (iTRAQ) and mass spectrometry to examine rare, primitive hematopoietic cells from patients with chronic myeloid leukemia. Mol Biotechnol 36, 81–9.PubMedCrossRefGoogle Scholar
  28. 28.
    Seshi, B. (2006) An integrated approach to mapping the proteome of the human bone marrow stromal cell. Proteomics 6, 5169–82.PubMedCrossRefGoogle Scholar
  29. 29.
    Thon, J. N., Schubert, P., Duguay, M., Serrano, K., Lin, S., Kast, J., and Devine, D. V. (2008) Comprehensive proteomic analysis of protein changes during platelet storage requires complementary proteomic approaches. Transfusion 48, 425–35.PubMedCrossRefGoogle Scholar
  30. 30.
    Wu, W. W., Wang, G. H., Baek, S. J., and Shen, R. F. (2006) Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDITOF/TOF. J Proteome Res 5, 651–58.PubMedCrossRefGoogle Scholar
  31. 31.
    Greengauz-Roberts, O., Stoppler, H., Nomura, S., Yamaguchi, H., Goldenring, J. R., Podolsky, R. H., Lee, J. R., and Dynan, W. S. (2005) Saturation labeling with cysteine-reactive cyanine fluorescent dyes provides increased sensitivity for protein expression profiling of laser-microdissected clinical specimens. Proteomics 5, 1746–57.PubMedCrossRefGoogle Scholar
  32. 32.
    Shadforth, I. P., Dunkley, T. P., Lilley, K. S., and Bessant, C. (2005) i-Tracker: for quantitative proteomics using iTRAQ. BMC Genomics 6, 145.PubMedCrossRefGoogle Scholar
  33. 33.
    Lin, W. T., Hung, W. N., Yian, Y. H., Wu, K. P., Han, C. L., Chen, Y. R., Chen, Y. J., Sung, T. Y., and Hsu, W. L. (2006) Multi-Q: a fully automated tool for multiplexed protein quantitation. J Proteome Res 5, 2328–38.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Peter Scherp
    • 1
  • Ginger Ku
    • 2
  • Liana Coleman
    • 2
  • Indu Kheterpal
    • 2
    Email author
  1. 1.Proteomics and Metabolomics Core, Pennington Biomedical Research CenterLouisiana State University SystemBaton RougeUSA
  2. 2.Protein Structural Biology, Pennington Biomedical Research CenterLouisiana State University SystemBaton RougeUSA

Personalised recommendations