Skip to main content
NCBI home page
NeuroRx logoLink to NeuroRx
. 2012 Sep 5;3(3):327–335. doi: 10.1016/j.nurx.2006.05.001

Two-dimensional protein electrophoresis: From molecular pathway discovery to biomarker discovery in neurological disorders

Leila H Choe 1, Brenda G Werner 1, Kelvin H Lee 1,✉
PMCID: PMC3593383  PMID: 16815216

Summary

Two-dimensional protein electrophoresis (2-DE) has undergone many technical improvements in the past 30 years, resulting in an analytical method that is unparalleled in the resolution of complex protein mixtures and capable of quantifying changes in protein expression from a wide variety of tissues and samples. The technique has been applied in many studies of neurologic disease to identify changes in spot patterns that correlate with disease. The true power of the technique emerges when it is coupled to state-of-the-art methods in mass spectrometry, which enable identification of the protein or proteins contained within a spot of interest on a 2-DE map. Investigators have successfully applied the technique to gain improved understanding of neurologic disease mechanisms in humans and in animal models and to discover biomarkers that are useful in the clinical setting. An important extension to these efforts that has not been realized thus far is the desire to profile changes in protein expression that result from therapy to help relate disease-modifying effects at the molecular level with clinical outcomes. Here we review the major advances in 2-DE methods and discuss specific examples of its application in the study of neurologic diseases.

Key Words: Proteomics, two-dimensional electrophoresis, neurological disorders, Alzheimer’s disease, Creutzfeldt-Jakob disease, biomarkers

References

  • 1.Gabor Miklos GL, Maleszka R. Rotein functions and biological contexts. Proteomics. 2001;1:169–178. doi: 10.1002/1615-9861(200101)1:1<30::AID-PROT30>3.0.CO;2-X. [DOI] [PubMed] [Google Scholar]
  • 2.Görg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology for proteomics. Proteomics. 2004;4:3665–3685. doi: 10.1002/pmic.200401031. [DOI] [PubMed] [Google Scholar]
  • 3.O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975;250:4007–4021. [PMC free article] [PubMed] [Google Scholar]
  • 4.Klose J. Rotein mapping by combined isoelectric focusing and electrophoresis of mouse tissues: a novel approach to testing for induced point mutations in mammals. Humangenetik. 1975;26:231–243. doi: 10.1007/BF00281458. [DOI] [PubMed] [Google Scholar]
  • 5.Ibarra F, Buhler JM. Rotein subunit mapping: a sensitive high resolution method. Anal Biochem. 1976;74:503–511. doi: 10.1016/0003-2697(76)90232-3. [DOI] [PubMed] [Google Scholar]
  • 6.Scheele GA. Two-dimensional gel analysis of soluble proteins: characterization of guinea pig exocrine pancreatic proteins. J Biol Chem. 1975;250:5375–5385. [PubMed] [Google Scholar]
  • 7.Shaw MM, Riederer BM. Sample preparation for two-dimensional gel electrophoresis. Proteomics. 2003;3:1408–1417. doi: 10.1002/pmic.200300471. [DOI] [PubMed] [Google Scholar]
  • 8.Rabilloud T. Solubilization of proteins for electrophoretic analysis. Electrophoresis. 1996;17:813–829. doi: 10.1002/elps.1150170503. [DOI] [PubMed] [Google Scholar]
  • 9.Huber LA, Pfaller K, Vietor I. Organelle proteomics: implications for subcellular fractionation in proteomics. Circ Res. 2003;92:962–968. doi: 10.1161/01.RES.0000071748.48338.25. [DOI] [PubMed] [Google Scholar]
  • 10.Zuo X, Speicher DW. A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis. Anal Biochem. 2000;284:266–278. doi: 10.1006/abio.2000.4714. [DOI] [PubMed] [Google Scholar]
  • 11.Björhall K, Miliotis T, Davidsson P. Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples. Proteomics. 2005;5:307–317. doi: 10.1002/pmic.200400900. [DOI] [PubMed] [Google Scholar]
  • 12.Granger J, Siddiqui J, Copeland S, et al. Albumin depletion of human plasma also removes low abundance proteins including the cytokines. Proteomics. 2005;5:4713–4718. doi: 10.1002/pmic.200401331. [DOI] [PubMed] [Google Scholar]
  • 13.Bjellqvist B, Ek K, Righetti PG, et al. Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J Biochem Biophys Methods. 1982;6:317–339. doi: 10.1016/0165-022X(82)90013-6. [DOI] [PubMed] [Google Scholar]
  • 14.Corbett JM, Dunn MJ, Posch A, et al. Positional reproducibility of protein spots in two-dimensional polyacrylamide gel electrophoresis using immobilized pH gradient isoelectric focusing in the first dimension: an interlaboratory comparison. Electrophoresis. 1994;15:1205–1211. doi: 10.1002/elps.11501501182. [DOI] [PubMed] [Google Scholar]
  • 15.Sanchez JC, Rouge V, Pisteur M, et al. Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis. 1997;18:324–327. doi: 10.1002/elps.1150180305. [DOI] [PubMed] [Google Scholar]
  • 16.Li ZB, Flint PW, Boluyt MO. Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics. Electrophoresis. 2005;26:3572–3585. doi: 10.1002/elps.200500104. [DOI] [PubMed] [Google Scholar]
  • 17.Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  • 18.Young D. Advantages of separation on “giant” two-dimensional gels for detection of physiologically relevant changes in the expression of protein gene-products. Clin Chem. 1984;30:2104–2106. [PubMed] [Google Scholar]
  • 19.Switzer RC, Merril CR, Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem. 1979;98:231–237. doi: 10.1016/0003-2697(79)90732-2. [DOI] [PubMed] [Google Scholar]
  • 20.Neuhoff V, Arold N, Taube D, et al. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis. 1988;9:255–262. doi: 10.1002/elps.1150090603. [DOI] [PubMed] [Google Scholar]
  • 21.Westermeier R, Marouga R. Rotein detection methods in proteomics research. Biosci Rep. 2005;25:19–31. doi: 10.1007/s10540-005-2845-1. [DOI] [PubMed] [Google Scholar]
  • 22.Berggren K, Chemokalskaya E, Steinberg TH, et al. Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex. Electrophoresis. 2000;21:2509–2521. doi: 10.1002/1522-2683(20000701)21:12<2509::AID-ELPS2509>3.0.CO;2-9. [DOI] [PubMed] [Google Scholar]
  • 23.Nishihara JC, Champion KM. Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain. Electrophoresis. 2002;23:2203–2215. doi: 10.1002/1522-2683(200207)23:14<2203::AID-ELPS2203>3.0.CO;2-H. [DOI] [PubMed] [Google Scholar]
  • 24.Luo L, Wirth PJ. Consecutive silver staining and autoradiography of35S and32P-labeled cellular proteins: applications for analysis of signal transducing pathways. Electrophoresis. 1993;14:127–136. doi: 10.1002/elps.1150140121. [DOI] [PubMed] [Google Scholar]
  • 25.Unlu M, Morgan ME, Minden JS. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis. 1997;18:2071–2077. doi: 10.1002/elps.1150181133. [DOI] [PubMed] [Google Scholar]
  • 26.Lescuyer P, Allard L, Zimmermann-Ivol CG, et al. Identification of postmortem cerebrospinal fluid proteins as potential biomarkers of ischemia and neurodegeneration. Proteomics. 2004;4:2234–2241. doi: 10.1002/pmic.200300822. [DOI] [PubMed] [Google Scholar]
  • 27.Schulenberg B, Goodman TN, Aggeler R, et al. Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry. Electrophoresis. 2004;25:2526–2532. doi: 10.1002/elps.200406007. [DOI] [PubMed] [Google Scholar]
  • 28.Hart C, Schulenberg B, Steinberg TH, et al. Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain. Electrophoresis. 2003;24:588–598. doi: 10.1002/elps.200390069. [DOI] [PubMed] [Google Scholar]
  • 29.Aksenov MY, Aksenova MV, Butterfield DA, et al. Protein oxidation in the brain in Alzheimer’s disease. Neuroscience. 2001;103:373–383. doi: 10.1016/S0306-4522(00)00580-7. [DOI] [PubMed] [Google Scholar]
  • 30.Castegna A, Thongboonkerd V, Klein JB, et al. Proteomic identification of nitrated proteins in Alzheimer’s disease brain. J Neurochem. 2003;85:1394–1401. doi: 10.1046/j.1471-4159.2003.01786.x. [DOI] [PubMed] [Google Scholar]
  • 31.Aebersold RH, Leavitt J, Saavedra RA, et al. Internal amino-acid sequence-analysis of proteins separated by one-dimensional or two-dimensional gel-electrophoresis afterin situ protease digestion on nitrocellulose. Proc Natl Acad Sci USA. 1987;84:6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Montine TJ, Woltjer RL, Pan C, et al. Liquid chromatography with tandem mass spectrometry-based proteomic discovery in aging and Alzheimer’s disease. NeuroRx. 2006;3:335–342. doi: 10.1016/j.nurx.2006.05.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Finehout EJ, Lee KH. An introduction to mass spectrometry applications in biological research. Biochem Mol Biol Educ. 2004;32:93–100. doi: 10.1002/bmb.2004.494032020331. [DOI] [PubMed] [Google Scholar]
  • 34.Finehout EJ, Lee KH. Comparison of automated in-gel digest methods for femtomole level samples. Electrophoresis. 2003;24:3508–3516. doi: 10.1002/elps.200305615. [DOI] [PubMed] [Google Scholar]
  • 35.Choe LH, Lee KH. Quantitative and qualitative measure of intralaboratory two-dimensional protein gel reproducibility and the effects of sample preparation, sample load, and image analysis. Electrophoresis. 2003;24:3500–3507. doi: 10.1002/elps.200305614. [DOI] [PubMed] [Google Scholar]
  • 36.Nishihara JC, Champion KM. Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain. Electrophoresis. 2002;23:2203–2215. doi: 10.1002/1522-2683(200207)23:14<2203::AID-ELPS2203>3.0.CO;2-H. [DOI] [PubMed] [Google Scholar]
  • 37.Molloy MP, Brzezinski EE, Hang J, et al. Overcoming technical variation and biological variation in quantitative proteomics. Proteomics. 2003;3:1912–1919. doi: 10.1002/pmic.200300534. [DOI] [PubMed] [Google Scholar]
  • 38.Lilley KS, Razzaq A, Dupree P. Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation. Curr Opin Chem Biol. 2001;6:46–50. doi: 10.1016/S1367-5931(01)00275-7. [DOI] [PubMed] [Google Scholar]
  • 39.Garbis S, Lubec G, Fountoulakis M. Limitations of current proteomics technologies. J Chromatogr A. 2005;1077:1–18. doi: 10.1016/j.chroma.2005.04.059. [DOI] [PubMed] [Google Scholar]
  • 40.Beranova-Giorgianni S. Proteome analysis by two-dimensional gel electrophoresis and mass spectrometry: strengths and limitations. Trends Anal Chem. 2003;22:273–281. doi: 10.1016/S0165-9936(03)00508-9. [DOI] [Google Scholar]
  • 41.Strohman R. Epigenesis: the missing beat in biotechnology. Biotechnology. 1994;12:156–164. doi: 10.1038/nbt0294-156. [DOI] [PubMed] [Google Scholar]
  • 42.Finehout EJ, Franck Z, Lee KH. Towards two-dimensional electrophoresis mapping of the cerebrospinal fluid proteome from a single individual. Electrophoresis. 2001;25:2564–2575. doi: 10.1002/elps.200406012. [DOI] [PubMed] [Google Scholar]
  • 43.Sanchez JC, Appel RD, Golaz O, et al. Inside SWISS-2D PAGE database. Electrophoresis. 1995;16:1131–1151. doi: 10.1002/elps.11501601190. [DOI] [PubMed] [Google Scholar]
  • 44.Tsuji T, Shimohama S. Analysis of the proteomic profiling of brain tissue in Alzheimer’s disease. Dis Markers. 2001;17:247–257. doi: 10.1155/2001/386284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Fountoulakis M. Application of proteomics technologies in the investigation of the brain. Mass Spectrom Rev. 2004;23:231–258. doi: 10.1002/mas.10075. [DOI] [PubMed] [Google Scholar]
  • 46.Vercauteren FGG, Bergeron JJM, Vandesande F, et al. Proteomic approaches in brain research and neuropharmacology. Eur J Pharmacol. 2004;500:385–398. doi: 10.1016/j.ejphar.2004.07.039. [DOI] [PubMed] [Google Scholar]
  • 47.Gong CX, Liu F, Grundke-Iqbal I, et al. Post-translational modifications of tau protein in Alzheimer’s disease. J Neural Transm. 2005;112:813–838. doi: 10.1007/s00702-004-0221-0. [DOI] [PubMed] [Google Scholar]
  • 48.Golde TE, Eckman CB, Younkin SC. Biochemical detection of A beta isoforms: implications for pathogenesis, diagnosis, and treatment of Alzheimer’s disease. Biochim Biophys Acta. 2000;1502:172–187. doi: 10.1016/S0925-4439(00)00043-0. [DOI] [PubMed] [Google Scholar]
  • 49.Kanninen K, Goldstein G, Auriola S, et al. Glycosylation changes in Alzheimer’s disease as revealed by a proteomic approach. Neurosci Lett. 2004;367:235–240. doi: 10.1016/j.neulet.2004.06.013. [DOI] [PubMed] [Google Scholar]
  • 50.Butterfield DA, Abdul MH, Newman S, Reed T. Redox proteomics in age-related neurodegenerative disorders or models thereof. NeuroRx. 2006;3:343–356. doi: 10.1016/j.nurx.2006.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Markesbery WR. Oxidative stress hypothesis in Alzheimer’s disease. Free Radic Biol Med. 1997;23:134–147. doi: 10.1016/S0891-5849(96)00629-6. [DOI] [PubMed] [Google Scholar]
  • 52.Castegna A, Aksenov M, Aksenova M, et al. Proteomic identification of oxidatively modified proteins in Alzheimer’s disease brain. Part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. Free Radic Biol Med. 2002;33:562–571. doi: 10.1016/S0891-5849(02)00914-0. [DOI] [PubMed] [Google Scholar]
  • 53.Cottrell BA, Galvan V, Banwait S, et al. A pilot proteomic study of amyloid precursor interactions in Alzheimer’s disease. Ann Neurol. 2005;58:277–289. doi: 10.1002/ana.20554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Tilleman K, van den Haute C, Geerts H, et al. Proteomics analysis of the neurodegeneration in the brain of tau transgenic mice. Proteomics. 2002;2:656–665. doi: 10.1002/1615-9861(200206)2:6<656::AID-PROT656>3.0.CO;2-Z. [DOI] [PubMed] [Google Scholar]
  • 55.David DC, Hauptmann S, Scherping I, et al. Proteomic and functional analyses reveal a mitochondrial dysfunction in P301L tau transgenic mice. J Biol Chem. 2005;280:23802–23814. doi: 10.1074/jbc.M500356200. [DOI] [PubMed] [Google Scholar]
  • 56.Harrington MG, Merril CR, Asher DM, et al. Abnormal proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease. N Engl J Med. 1986;315:279–283. doi: 10.1056/NEJM198607313150502. [DOI] [PubMed] [Google Scholar]
  • 57.Hsich G, Kenney K, Gibbs CJ, et al. The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform en-cephalopathies. N Engl J Med. 1996;335:924–930. doi: 10.1056/NEJM199609263351303. [DOI] [PubMed] [Google Scholar]
  • 58.Green AJE, Thompson EJ, Stewart GE, et al. Use of 14-3-3 and other brain-specific proteins in CSF in the diagnosis of variant Creutzfeldt-Jakob disease. J Neurol Neurosurg Psychiatry. 2001;70:744–748. doi: 10.1136/jnnp.70.6.744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.van Everbroeck B, Boons J, Cras P. Cerebrospinal fluid biomarkers in Creutzfeldt-Jakob disease. Clin Neurol Neurosurg. 2005;107:355–360. doi: 10.1016/j.clineuro.2004.12.002. [DOI] [PubMed] [Google Scholar]
  • 60.World Health Organization Human transmissible spongiform en-cephalopathies. Wkly Epidemol Rec. 1998;73:361–365. [Google Scholar]
  • 61.Choe LH, Green A, Knight RSG, et al. Apolipoprotein E and other cerebrospinal fluid proteins differentiate antemortem variant Creutzfeldt-Jakob disease from antemortem sporadic Creutzfeldt-Jakob disease. Electrophoresis. 2002;23:2242–2246. doi: 10.1002/1522-2683(200207)23:14<2242::AID-ELPS2242>3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]
  • 62.Davidsson P, Westman-Brinkmalm A, Nilsson CL, et al. Proteome analysis of cerebrospinal fluid proteins in Alzheimer patients. NeuroReport. 2002;13:611–615. doi: 10.1097/00001756-200204160-00015. [DOI] [PubMed] [Google Scholar]
  • 63.Davidsson P, Sjogren M, Andreasen N, et al. Studies of the pathophysiological mechanisms in frontotemporal dementia by proteome analysis of CSF proteins. Mol Brain Res. 2002;109:128–133. doi: 10.1016/S0169-328X(02)00549-1. [DOI] [PubMed] [Google Scholar]
  • 64.Finehout EJ, Franck Z, Lee KH. Complement protein isoforms in CSF as possible biomarkers for neurodegenerative disease. Dis Markers. 2005;21:93–101. doi: 10.1155/2005/806573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Lescuyer P, Gandini A, Burkhard PR, et al. Prostaglandin D2 synthase and its post-translational modifications in neurological disorders. Electrophoresis. 2005;26:4563–4570. doi: 10.1002/elps.200500292. [DOI] [PubMed] [Google Scholar]
  • 66.Scott RB. Extraneuronal manifestations of Alzheimer’s disease. J Am Geriatr Soc. 1993;41:268–276. doi: 10.1111/j.1532-5415.1993.tb06704.x. [DOI] [PubMed] [Google Scholar]
  • 67.Choi J, Malakowsky CA, Talent JM, et al. Identification of oxidized plasma proteins in Alzheimer’s disease. Biochem Biophys Res Commun. 2002;293:1566–1570. doi: 10.1016/S0006-291X(02)00420-5. [DOI] [PubMed] [Google Scholar]
  • 68.Yu HL, Chertkow HM, Bergman H, et al. Aberrant profiles of native and oxidized glycoproteins in Alzheimer plasma. Proteomics. 2003;3:2240–2248. doi: 10.1002/pmic.200300475. [DOI] [PubMed] [Google Scholar]
  • 69.Mattila KM, Frey H. Two-dimensional analysis of qualitative and quantitative changes in blood cell proteins in Alzheimer’s disease: search for extraneuronal markers. Appl Theor Electrophor. 1995;4:189–196. [PubMed] [Google Scholar]
  • 70.Jabbour W, Pouplard-Barthelaix A, Houlgatte R, et al. Abnormal expression of actin in lymphocytes of Alzheimer’s disease and Down’s syndrome patients. J Neuroimmunol. 1992;38:199–208. doi: 10.1016/0165-5728(92)90013-B. [DOI] [PubMed] [Google Scholar]
  • 71.Rohlff C. Proteomics in molecular medicine: applications in central nervous systems disorders. Electrophoresis. 2000;21:1227–1234. doi: 10.1002/(SICI)1522-2683(20000401)21:6<1227::AID-ELPS1227>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  • 72.D’Ascenzo M, Relkin NR, Lee KH. Alzheimer’s disease cerebrospinal fluid biomarker discovery: a proteomics approach. Curr Opin Mol Ther. 2005;7:557–564. [PubMed] [Google Scholar]
  • 73.Listgarten J, Emili A. Practical proteomic biomarker discovery: taking a step back to leap forward. Drug Disc Today. 2005;10:1697–1702. doi: 10.1016/S1359-6446(05)03645-7. [DOI] [PubMed] [Google Scholar]

Articles from NeuroRx are provided here courtesy of Am. Soc. for Experimental NeuroTherapeutics

RESOURCES