The application of DIGE-based proteomics to renal physiology.
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.
Hoorn
Ewout J
EJ
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md. 20892, USA.
Hoffert
Jason D
JD
Knepper
Mark A
MA
D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007685:Kidney Tubules, Collecting / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification; D040901:Proteomics; D017483:Receptors, Vasopressin / Q000378:metabolism; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D049490:Systems Biology; D014882:Water-Electrolyte Balance
D051399:Aquaporin 2; D008565:Membrane Proteins; D017483:Receptors, Vasopressin