Proteomics is seeing increasing use as a means of identifying new mechanistic hypotheses in physiology. Proteomics based on two-dimensional electrophoresis (2-DE) has recently been optimized with the development of Difference Gel Electrophoresis (DIGE). In DIGE-based proteomics, the experimental and control samples are derivatized with different fluorophores and are run in the same gel, thereby minimizing technical variation. DIGE is currently one of the few techniques to perform quantitative proteomics, generating a statistical output to differences in protein abundances. In this review, we discuss the principles of DIGE-based proteomics, including sample preparation, 2-DE, statistical analysis of 2D-gels, and mass spectrometry. Strengths and weaknesses of DIGE are discussed, including possible solutions to overcome certain limitations, such as the identification of low abundance and integral membrane proteins. In addition, we provide a brief synopsis of our recent experiments in which DIGE-based proteomics was applied to study vasopressin signaling in the renal collecting duct. Finally, we illustrate how quantification based on the DIGE approach combined with bioinformatics may facilitate the study of systems biology of the kidney.

Keywords:
Bioinformatics, Difference Gel Electrophoresis, Mass spectrometry, Pathways analysis, Systems biology, Vasopressin
1.
Knepper MA: Proteomics and the kidney. J Am Soc Nephrol 2002;13:1398–1408.
2.
Aebersold R, Mann M: Mass spectrometry-based proteomics. Nature 2003;422:198–207.
3.
Hoorn EJ, Hoffert JD, Knepper MA: Combined proteomics and pathways analysis of collecting duct reveals a protein regulatory network activated in vasopressin escape. J Am Soc Nephrol 2005;16:2852–2863.
4.
Steen H, Pandey A: Proteomics goes quantitative: measuring protein abundance. Trends Biotechnol 2002;20:361–364.
5.
VanBogelen RA, Molloy MP: Exploring the proteome: reviving emphasis on quantitative protein profiling. Proteomics 2003;3:1833–1834.
6.
Molloy MP, Brzezinski EE, Hang J, McDowell MT, VanBogelen RA: Overcoming technical variation and biological variation in quantitative proteomics. Proteomics 2003;3:1912–1929.
7.
Gorg A, Weiss W, Dunn MJ: Current two-dimensional electrophoresis technology for proteomics. Proteomics 2004;4:3665–3685.
8.
Unlu M, Morgan ME, Minden JS: Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 1997;18:2071–2077.
9.
de Graauw M, Tijdens I, Cramer R, Corless S, Timms JF, van de Water B: Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis. J Biol Chem 2005;280:29885–29898.
10.
Sharma K, Lee S, Han S, Francos B, McCue P, Wassell R, et al: Two-dimensional fluorescence difference gel electrophoresis analysis of the urine proteome in human diabetic nephropathy. Proteomics 2005;5:2648–2655.
11.
Curthoys NP, Taylor L, Hoffert JD, Knepper MA: Proteomic analysis of the adaptive response of rat renal proximal tubules to metabolic acidosis. In preparation.
12.
van Balkom BW, Hoffert JD, Chou CL, Knepper MA: Proteomic analysis of long-term vasopressin action in the inner medullary collecting duct of the Brattleboro rat. Am J Physiol Renal Physiol 2004;286:F216–F224.
13.
Hoffert JD, van Balkom BW, Chou CL, Knepper MA: Application of difference gel electrophoresis to the identification of inner medullary collecting duct proteins. Am J Physiol Renal Physiol 2004;286:F170–F179.
14.
Yates JR 3rd, Gilchrist A, Howell KE, Bergeron JJ: Proteomics of organelles and large cellular structures. Nat Rev Mol Cell Biol 2005;6:702–714.
15.
Petricoin EF, Liotta LA: SELDI-TOF-based serum proteomic pattern diagnostics for early detection of cancer. Curr Opin Biotechnol 2004;15:24–30.
16.
Pixton KL: Is it functional? Report on the first BSPR/EBI meeting on functional proteomics. Proteomics 2004;4:3762–3764.
17.
Hoorn EJ, Pisitkun T, Zietse R, Gross P, Frokiaer J, Wang NS, et al: Prospects for urinary proteomics: exosomes as a source of urinary biomarkers. Nephrology (Carlton) 2005;10:283–290.
18.
Lilley KS, Friedman DB: All about DIGE: quantification technology for differential-display 2D-gel proteomics. Expert Rev Proteomics 2004;1:401–409.
19.
Alban A, David SO, Bjorkesten L, Andersson C, Sloge E, Lewis S, et al: A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 2003;3:36–44.
20.
Shaw J, Rowlinson R, Nickson J, Stone T, Sweet A, Williams K, et al: Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes. Proteomics 2003;3:1181–1195.
21.
Stasyk T, Huber LA: Zooming in: fractionation strategies in proteomics. Proteomics 2004;4:3704–3716.
22.
Oda Y, Nagasu T, Chait BT: Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat Biotechnol 2001;19:379–382.
23.
Klein E, Klein JB, Thongboonkerd V: Two-dimensional gel electrophoresis: a fundamental tool for expression proteomics studies. Contrib Nephrol 2004;141:25–39.
24.
Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, et al: The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000;21:1037–1053.
25.
Fodor IK, Nelson DO, Alegria-Hartman M, Robbins K, Langlois RG, Turteltaub KW, et al: Statistical challenges in the analysis of two-dimensional difference gel electrophoresis experiments using DeCyderTM. Bioinformatics 2005;21:3733–3740.
26.
Lonstedt I, Speed TP: Replicated micro-array data. Stat Sin 2002;12:31–46.
27.
Bland JM, Altman DG: Multiple significance tests: the Bonferroni method. BMJ 1995;310:170.
28.
Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I: Controlling the false discovery rate in behavior genetics research. Behav Brain Res 2001;125:279–284.
29.
Kreil DP, Karp NA, Lilley KS: DNA microarray normalization methods can remove bias from differential protein expression analysis of 2D difference gel electrophoresis results. Bioinformatics 2004;20:2026–2034.
30.
Arthur JM: Proteomics. Curr Opin Nephrol Hypertens 2003;12:423–430.
31.
Nielsen S, Frokiaer J, Marples D, Kwon TH, Agre P, Knepper MA: Aquaporins in the kidney: from molecules to medicine. Physiol Rev 2002;82:205–244.
32.
Stokes JB, Grupp C, Kinne RK: Purification of rat papillary collecting duct cells: functional and metabolic assessment. Am J Physiol 1987;253(Pt 2):F251–F262.
33.
Laycock JF: Review: the Brattleboro rat with hereditary hypothalamic diabetes insipidus as an ideal experimental model. Lab Anim 1976;10:261–270.
34.
Adrogue HJ, Madias NE: Hyponatremia. N Engl J Med 2000;342:1581–1589.
35.
Jaenike JR, Waterhouse C: The renal response to sustained administration of vasopressin and water in man. J Clin Endocrinol Metab 1961;21:231–242.
36.
Schwartz WB, Bennett W, Curelop S, Bartter FC: A syndrome of renal sodium loss and hyponatremia probably resulting from inappropriate secretion of antidiuretic hormone. 1957. J Am Soc Nephrol 2001;12:2860–2870.
37.
Qin S, Ferdinand AS, Richie JP, O’Leary MP, Mok SC, Liu BC: Chromatofocusing fractionation and two-dimensional difference gel electrophoresis for low abundance serum proteins. Proteomics 2005;5:3183–3192.
38.
Xiong S, Ding Q, Zhao Z, Chen W, Wang G, Liu S: A new method to improve sensitivity and resolution in matrix-assisted laser desorption/ionization time of flight mass spectrometry. Proteomics 2003;3:265–272.
39.
Luche S, Santoni V, Rabilloud T: Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. Proteomics 2003;3:249–253.
40.
Santoni V, Molloy M, Rabilloud T: Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000;21:1054–1070.
41.
Macfarlane DE: Two dimensional benzyldimethyl-n-hexadecylammonium chloride-sodium dodecyl sulfate preparative polyacrylamide gel electrophoresis: a high capacity high resolution technique for the purification of proteins from complex mixtures. Anal Biochem 1989;176:457–463.
42.
Hartinger J, Stenius K, Hogemann D, Jahn R: 16-BAC/SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins. Anal Biochem 1996;240:126–133.
43.
Bunai K, Yamane K: Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives. J Chromatogr B Analyt Technol Biomed Life Sci 2005;815:227–236.
44.
Zahedi RP, Meisinger C, Sickmann A: Two-dimensional benzyldimethyl-n-hexadecylammonium chloride/SDS-PAGE for membrane proteomics. Proteomics 2005;5:3581–3588.
45.
Knepper MA, Masilamani S: Targeted proteomics in the kidney using ensembles of antibodies. Acta Physiol Scand 2001;173:11–21.
46.
Westerhoff HV, Palsson BO: The evolution of molecular biology into systems biology. Nat Biotechnol 2004;22:1249–1252.
47.
Aderem A: Systems biology: its practice and challenges. Cell 2005;121:511–513.
48.
Kalume DE, Molina H, Pandey A: Tackling the phosphoproteome: tools and strategies. Curr Opin Chem Biol 2003;7:64–69.
49.
Liu ET: Systems biology, integrative biology, predictive biology. Cell 2005;121:505–506.
50.
Siripurapu V, Meth J, Kobayashi N, Hamaguchi M: DBC2 significantly influences cell-cycle, apoptosis, cytoskeleton and membrane-trafficking pathways. J Mol Biol 2005;346:83–89.
51.
Calvano SE, Xiao W, Richards DR, Felciano RM, Baker HV, Cho RJ, et al: A network-based analysis of systemic inflammation in humans. Nature 2005;437:1032–1037.
52.
Legato J, Knepper MA, Star RA, Mejia R: Database for renal collecting duct regulatory and transporter proteins. Physiol Genomics 2003;13:179–181.
53.
Papin JA, Hunter T, Palsson BO, Subramaniam S: Reconstruction of cellular signalling networks and analysis of their properties. Nat Rev Mol Cell Biol 2005;6:99–111.
54.
Sachs K, Perez O, Pe’er D, Lauffenburger DA, Nolan GP: Causal protein-signaling networks derived from multiparameter single-cell data. Science 2005;308:523–529.
55.
Moraru, II, Loew LM: Intracellular signaling: spatial and temporal control. Physiology (Bethesda) 2005;20:169–179.
56.
Hood L, Heath JR, Phelps ME, Lin B: Systems biology and new technologies enable predictive and preventative medicine. Science 2004;306:640–643.
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