Constitutive and inducible stress proteins dominate the proteome of the murine inner medullary collecting duct-3 (mIMCD3) cell line.

A proteome map of the most abundant proteins in the murine inner medullary collecting duct (mIMCD3) cell line was generated by 2-dimensional gel electrophoresis (2D-GE) combined with MALDI-TOF/TOF mass spectrometry. The 2-D model map identifies 77 distinct constitutive proteins and a total of 86 spots including isoforms. Protein identification was based on both peptide mass fingerprinting (MS) and peptide fragmentation (MS/MS) data. High confidence Mascot scores were obtained in the database search, due to the high quality and the number of MS/MS spectra which provided matching sequence information to the database. A functional classification of the identified proteins showed that a high proportion were stress proteins, such as heat shock proteins and proteins with anti-oxidant activity. Other proteins identified were involved in cytoskeletal maintenance, metabolism and energy generation, as well as in translation, transcription, RNA processing and other cell cycle processes. Exposure of the mIMCD3 cells to hyperosmotic stress using 600 mOsmol/kg NaCl or Urea or 700 mOsmol/kg NaCl-Urea (50:50) resulted in the greatest proteome upregulation in 700 mosM NaCl-Urea and the greatest downregulation in 600 mosM NaCl. Several proteins with molecular chaperone function were induced, such as alpha-B crystallin, two Hsp70 isoforms, the osmotic stress protein (Osp94), as well as aldose reductase. Additional isoforms of the translation elongation factors Eef2 and Eef1a1 were induced. Characterization of the phosphoproteome of mIMCD3 cells with a phosphoprotein-specific stain showed a significant proportion of the proteome was phosphorylated. Additionally, exposure of mIMCD3 cells to 600 mOsmol/kg NaCl hyperosmotic stress resulted in a 1.8-fold higher phosphorylation level of the most acidic isoform of the heat shock protein Hsp27 compared to its phosphorylation level under iso-osmotic conditions.