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Am J Physiol Renal Physiol (September 9, 2003). doi:10.1152/ajprenal.00223.2003
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Submitted on June 13, 2003
Accepted on September 2, 2003

Application of Difference Gel Electrophoresis (DIGE) to the Identification of Inner Medullary Collecting Duct Proteins

Jason D. Hoffert1, Bas W.M. van Balkom1, Chung-Lin Chou1, and Mark A. Knepper1*

1 Laboratory of Kidney and Electrolyte Metabolism, NHLBI, National Institutes of Health, Bethesda, MD, USA

* To whom correspondence should be addressed. E-mail: knep{at}helix.nih.gov.

In this study, we present a standardized approach to purification of native inner medullary collecting duct (IMCD) cells from rat kidney for proteomic analysis and apply the approach to identification of abundant proteins utilizing two-dimensional difference gel electrophoresis (DIGE) coupled with MALDI-TOF mass spectrometry. Fractionation of inner medullary cell suspensions by low-speed centrifugation gave a highly purified IMCD cell fraction in which aquaporin-2 was enriched 10-fold. When DIGE was initially applied to rat inner medullas fractionated into IMCD cells (labeled with Cy3) and non-IMCD cells (labeled with Cy5), we identified 50 highly abundant proteins expressed in the IMCD cells. These proteins identifiable without subcellular fractionation, included chiefly enzymes, structural proteins, and signaling intermediates. An additional 35 proteins were found predominantly in the non-IMCD cell types. Proteins that were highly enriched in the IMCD fraction included cytokeratin 8, cytokeratin 18, transglutaminase II, aminopeptidase B, T-plastin, HSP27, HSP70, and lactate dehydrogenase A. Semiquantitative immunoblotting and immunohistochemistry confirmed relative expression levels and distribution of selected proteins. An additional 40 IMCD proteins were identified in separate experiments aimed at further enrichment of proteins through optimization of sample loading. These studies document the applicability of a standardized approach to purification of IMCD cells for proteomic analysis of IMCD proteins and demonstrate the feasibility of large scale identification of proteins in the native IMCD cell.




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