Skip to main content
SpringerLink
Log in
SpringerLink
Log in

Elucidating Cellular Metabolism and Protein Difference Data from DIGE Proteomics Experiments Using Enzyme Assays

  • Protocol
  • First Online:
Difference Gel Electrophoresis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1664))

Abstract

Assays for measuring enzyme activity can be useful tools for proteomics applications. Enzyme testing can be performed to validate an experimental system prior to a Difference Gel Electrophoresis (DIGE) proteomic experiment and can also be utilized as an integral part of multifaceted experiment in conjunction with DIGE. Data from enzyme tests can be used to corroborate results of DIGE proteomic experiments where an enzyme or enzymes are demonstrated by DIGE to be differentially expressed. Enzyme testing can also be utilized to support data from DIGE experiments that demonstrate metabolic changes in a biological system. The different types of enzyme assays that can be performed in conjunction with DIGE experiments are reviewed alongside a discussion of experimental approaches for designing enzyme assays.

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 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Sandberg AS, Buschmann V, Kapusta P et al (2016) Use of time-resolved fluorescence to improve sensitivity and dynamic range of gel-based proteomics. Anal Chem 88(6):3067–3074. doi:10.1021/acs.analchem.5b03805

    Article  CAS  PubMed  Google Scholar 

  2. Hurd TR, Prime TA, Harbour ME et al (2007) Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling. J Biol Chem 282(30):22040–22051. doi:10.1074/jbc.M703591200

    Article  CAS  PubMed  Google Scholar 

  3. Morak M, Schmidinger H, Krempl P et al (2009) Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities. J Lipid Res 50(7):1281–1292. doi:10.1194/jlr.M800566-JLR200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. O’Connell K, Ohlendieck K (2009) Proteomic DIGE analysis of the mitochondria-enriched fraction from aged rat skeletal muscle. Proteomics 9(24):5509–5524. doi:10.1002/pmic.200900472

    Article  PubMed  Google Scholar 

  5. Bentaib A, De Tullio P, Chneiweiss H et al (2014) Metabolic reprogramming in transformed mouse cortical astrocytes: a proteomic study. J Proteome 113:292–314. doi:10.1016/j.jprot.2014.09.019

    Article  Google Scholar 

  6. Faure C, Morio B, Chafey P et al (2013) Citrulline enhances myofibrillar constituents expression of skeletal muscle and induces a switch in muscle energy metabolism in malnourished aged rats. Proteomics 13(14):2191–2201. doi:10.1002/pmic.201200262

    Article  CAS  PubMed  Google Scholar 

  7. Lubec G, Afjehi-Sadat L, Yang JW, John JPP (2005) Searching for hypothetical proteins: theory and practice based upon original data and literature. Prog Neurobiol 77(1–2):90–127. doi:10.1016/j.pneurobio.2005.10.001

    Article  CAS  PubMed  Google Scholar 

  8. Afjehi-Sadat L, Lubec G (2007) Identification of enzymes and activity from two-dimensional gel electrophoresis. Nat Protoc 2(10):2318–2324. doi:10.1038/nprot.2007.317

    Article  CAS  PubMed  Google Scholar 

  9. Boone CHT, Grove RA, Adamcova D et al (2016) Revealing oxidative damage to enzymes of carbohydrate metabolism in yeast: an integration of 2D DIGE, quantitative proteomics, and bioinformatics. Proteomics 16(13):1889–1903. doi:10.1002/pmic.201500546

    Article  CAS  PubMed  Google Scholar 

  10. Mandili G, Alchera E, Merlin S et al (2015) Mouse hepatocytes and LSEC proteome reveal novel mechanisms of ischemia/reperfusion damage and protection by A2aR stimulation. J Hepatol 62(3):573–580. doi:10.1016/j.jhep.2014.10.007

    Article  CAS  PubMed  Google Scholar 

  11. Fekkar A, Balloy V, Pionneau C et al (2012) Secretome of human bronchial epithelial cells in response to the fungal pathogen Aspergillus fumigatus analyzed by differential in-gel electrophoresis. J Infect Dis 205(7):1163–1172. doi:10.1093/infdis/jis031

    Article  CAS  PubMed  Google Scholar 

  12. Overgaard J, Sørensen JG, Com E, Colinet H (2014) The rapid cold hardening response of Drosophila melanogaster: complex regulation across different levels of biological organization. J Insect Physiol 62(1):46–53. doi:10.1016/j.jinsphys.2014.01.009

    Article  CAS  PubMed  Google Scholar 

  13. Winkler J, Stessl M, Amartey J, Noe CR (2010) Off-target effects related to the phosphorothioate modification of nucleic acids. ChemMedChem 5(8):1344–1352. doi:10.1002/cmdc.201000156

    Article  CAS  PubMed  Google Scholar 

  14. Fanjul-fernández M, Folgueras AR, Fueyo A et al (2013) Matrix metalloproteinase Mmp-1a is dispensable for normal growth and fertility in mice and promotes lung cancer progression by modulating inflammatory responses. J Biol Chem 288(20):14647–14656. doi:10.1074/jbc.M112.439893

    Article  PubMed  PubMed Central  Google Scholar 

  15. Wendelboe-Nelson C, Morris PC (2012) Proteins linked to drought tolerance revealed by DIGE analysis of drought resistant and susceptible barley varieties. Proteomics 12(22):3374–3385. doi:10.1002/pmic.201200154

    Article  CAS  PubMed  Google Scholar 

  16. He C, Gao G, Zhang J et al (2016) Proteome profiling reveals insights into cold-tolerant growth in sea buckthorn. Proteome Sci 14(1):14. doi:10.1186/s12953-016-0103-z

    Article  PubMed  PubMed Central  Google Scholar 

  17. Schröder C, Matthies A, Engst W et al (2013) Identification and expression of genes involved in the conversion of daidzein and genistein by the equol-forming bacterium slackia isoflavoniconvertens. Appl Environ Microbiol 79(11):3494–3502. doi:10.1128/AEM.03693-12

    Article  PubMed  PubMed Central  Google Scholar 

  18. Jindal HK, Merchant E, Balschi JA et al (2012) Proteomic analyses of transgenic LQT1 and LQT2 rabbit hearts elucidate an increase in expression and activity of energy producing enzymes. J Proteome 75(17):5254–5265. doi:10.1016/j.jprot.2012.06.034

    Article  CAS  Google Scholar 

  19. Lennicke C, Rahn J, Kipp AP et al (2017) Individual effects of different selenocompounds on the hepatic proteome and energy metabolism of mice. Biochim Biophys Acta 1861(1 Pt A):3323–3334. doi:10.1016/j.bbagen.2016.08.015

    Article  CAS  PubMed  Google Scholar 

  20. He CY, Zhang GY, Zhang JG et al (2016) Physiological, biochemical, and proteome profiling reveals key pathways underlying the drought stress responses of Hippophae rhamnoides. Proteomics 16(20):2688–2697. doi:10.1002/pmic.201600160

    Article  CAS  PubMed  Google Scholar 

  21. Menazza S, Wong R, Nguyen T et al (2013) CypD−/− hearts have altered levels of proteins involved in Krebs cycle, branch chain amino acid degradation and pyruvate metabolism. J Mol Cell Cardiol 56:81–90. doi:10.1016/j.yjmcc.2012.12.004

    Article  CAS  PubMed  Google Scholar 

  22. Pieper R, Martin L, Schunter N et al (2015) Impact of high dietary zinc on zinc accumulation, enzyme activity and proteomic profiles in the pancreas of piglets. J Trace Elem Med Biol 30:30–36. doi:10.1016/j.jtemb.2015.01.008

    Article  CAS  PubMed  Google Scholar 

  23. Li X, Jin Z, Gao F et al (2014) Comparative proteomic analysis of Dan’er malts produced from distinct malting processes by two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE). J Agric Food Chem 62:9310–9316. doi:10.1021/jf5030483

    Article  CAS  PubMed  Google Scholar 

  24. García-Lorenzo A, Rodríguez-Piñeiro AM, Rodríguez-Berrocal FJ et al (2012) Changes on the Caco-2 secretome through differentiation analyzed by 2-D differential in-gel electrophoresis (DIGE). Int J Mol Sci 13(11):14401–14420. doi:10.3390/ijms131114401

    Article  PubMed  PubMed Central  Google Scholar 

  25. Marín-Buera L, García-Bartolomé A, Morán M et al (2015) Differential proteomic profiling unveils new molecular mechanisms associated with mitochondrial complex III deficiency. J Proteome 113:38–56. doi:10.1016/j.jprot.2014.09.007

    Article  Google Scholar 

  26. Gutiérrez-Sánchez G, Atwood J, Kolli VSK et al (2012) Initial proteome analysis of caffeine-induced proteins in Aspergillus tamarii using two-dimensional fluorescence difference gel electrophoresis. Appl Biochem Biotechnol 166(8):2064–2077. doi:10.1007/s12010-012-9634-7

    Article  PubMed  Google Scholar 

  27. Ray S, Patel SK, Venkatesh A et al (2016) Clinicopathological analysis and multipronged quantitative proteomics reveal oxidative stress and cytoskeletal proteins as possible markers for severe vivax malaria. Sci Rep 6:24557. doi:10.1038/srep24557

    Article  PubMed  PubMed Central  Google Scholar 

  28. Daneshvar H, Wyllie S, Phillips S et al (2012) Comparative proteomics profiling of a gentamicin-attenuated Leishmania infantum cell line identifies key changes in parasite thiol-redox metabolism. J Proteome 75(5):1463–1471. doi:10.1016/j.jprot.2011.11.018

    Article  CAS  Google Scholar 

  29. Drüppel K, Hensler M, Trautwein K et al (2014) Pathways and substrate-specific regulation of amino acid degradation in Phaeobacter inhibensDSM 17395 (archetype of the marine Roseobacter clade). Environ Microbiol 16(1):218–238. doi:10.1111/1462-2920.12276

    Article  PubMed  Google Scholar 

  30. Jia H, Shao M, He Y et al (2015) Proteome dynamics and physiological responses to short-term salt stress in Brassica napus leaves. PLoS One 10(12). doi:10.1371/journal.pone.0144808

  31. von Löhneysen K, Scott TM, Soldau K et al (2012) Assessment of the red cell proteome of young patients with unexplained hemolytic anemia by two-dimensional differential in-gel electrophoresis (DIGE). PLoS One 7(4). doi:10.1371/journal.pone.0034237

  32. De Palma S, Leone R, Grumati P et al (2013) Changes in muscle cell metabolism and mechanotransduction are associated with myopathic phenotype in a mouse model of collagen VI deficiency. PLoS One 8(2). doi:10.1371/journal.pone.0056716

  33. Capitanio D, Vasso M, Ratti A et al (2012) Molecular signatures of amyotrophic lateral sclerosis disease progression in hind and forelimb muscles of an SOD1(G93A) mouse model. Antioxid Redox Signal 17(10):1333–1350. doi:10.1089/ars.2012.4524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Dorts J, Kestemont P, Thézenas ML et al (2014) Effects of cadmium exposure on the gill proteome of Cottus gobio: modulatory effects of prior thermal acclimation. Aquat Toxicol 154:87–96. doi:10.1016/j.aquatox.2014.04.030

    Article  CAS  PubMed  Google Scholar 

  35. Kube M, Chernikova TN, Al-Ramahi Y et al (2013) Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica. Nat Commun 4:2156. doi:10.1038/ncomms3156

    Article  PubMed  PubMed Central  Google Scholar 

  36. Junghare M, Spiteller D, Schink B (2016) Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01. Environ Microbiol 18(9):3175–3188. doi:10.1111/1462-2920.13447

    Article  CAS  PubMed  Google Scholar 

  37. Bisswanger H (2014) Enzyme assays. Perspect Sci 1(1–6):41–55. doi:10.1016/j.pisc.2014.02.005

    Article  Google Scholar 

  38. Trivedi M, Laurence J, Siahaan T (2009) The role of thiols and disulfides on protein stability. Curr Protein Pept Sci 10(6):614–625. doi:10.2174/138920309789630534

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Fágáin CÓ (1995) Understanding and increasing protein stability. Biochim Biophys Acta 1252(1):1–14. doi:10.1016/0167-4838(95)00133-F

    Article  PubMed  Google Scholar 

  40. Janssen AJM, Trijbels FJM, Sengers RCA et al (2007) Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts. Clin Chem 53(4):729–734. doi:10.1373/clinchem.2006.078873

    Article  CAS  PubMed  Google Scholar 

  41. Aehle W (2007) Enzymes in industry: production and applications. Wiley, Weinheim. doi:10.1002/9783527617098

    Book  Google Scholar 

  42. Linn TC, Pettit FH, Reed LJ (1969) Alpha-keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation. Proc Natl Acad Sci U S A 62:234–241. doi:10.1073/pnas.62.1.234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Monaghan Biosciences, Tyholland, Co. Monaghan, Ireland

    Andrew Dowd

Authors
  1. Andrew Dowd
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andrew Dowd .

Editor information

Editors and Affiliations

  1. Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland

    Kay Ohlendieck

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Dowd, A. (2018). Elucidating Cellular Metabolism and Protein Difference Data from DIGE Proteomics Experiments Using Enzyme Assays. In: Ohlendieck, K. (eds) Difference Gel Electrophoresis. Methods in Molecular Biology, vol 1664. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7268-5_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7268-5_20

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7267-8

  • Online ISBN: 978-1-4939-7268-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation