sodium-dodecyl-sulfate and Polycystic-Kidney--Autosomal-Dominant

Toward multiplexed, comprehensive, and robust quantitation of the membrane proteome, we report a strategy combining gel-assisted digestion, iTRAQ (isobaric tags for relative and absolute quantitation) labeling, and LC-MS/MS. Quantitation of four independently purified membrane fractions from HeLa cells gave high accuracy (<8% error) and precision (<12% relative S.D.), demonstrating a high degree of consistency and reproducibility of this quantitation platform. Under stringent identification criteria (false discovery rate = 0%), the strategy efficiently quantified membrane proteins; as many as 520 proteins (91%) were membrane proteins, each quantified based on an average of 14.1 peptides per integral membrane protein. In addition to significant improvements in signal intensity for most quantified proteins, most remarkably, topological analysis revealed that the biggest improvement was achieved in detection of transmembrane peptides from integral membrane proteins with up to 19 transmembrane helices. To the best of our knowledge, this level of coverage exceeds that achieved previously using MS and provides superior quantitation accuracy compared with other methods. We applied this approach to the first proteomics delineation of phenotypic expression in a mouse model of autosomal dominant polycystic kidney disease (ADPKD). By characterizing kidney cell plasma membrane from wild-type versus PKD1 knock-out mice, 791 proteins were quantified, and 67 and 37 proteins showed > or =2-fold up-regulation and down-regulation, respectively. Some of these differentially expressed membrane proteins are involved in the mechanisms underlying major abnormalities in ADPKD, including epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, ion and fluid secretion, and membrane protein polarity. Among these proteins, targeting therapeutics to certain transporters/receptors, such as epidermal growth factor receptor, has proven effective in preclinical studies of ADPKD; others are known drug targets in various diseases. Our method demonstrates how comparative membrane proteomics can provide insight into the molecular mechanisms underlying ADPKD and the identification of potential drug targets, which may lead to new therapeutic opportunities to prevent or retard the disease.

Topics: Amino Acid Sequence; Animals; Cell Fractionation; Chromatography, Ion Exchange; HeLa Cells; Humans; Isotope Labeling; Kidney; Membrane Proteins; Mice; Models, Biological; Molecular Sequence Data; Polycystic Kidney, Autosomal Dominant; Protein Transport; Proteome; Proteomics; Reproducibility of Results; Sodium Dodecyl Sulfate; Sodium-Potassium-Exchanging ATPase; Subcellular Fractions

Cyst fluid samples obtained from eight patients with autosomal dominant polycystic kidney disease (ADPKD) were analysed for the presence of the basement membrane component laminin and its breakdown products, using ELISA and immunoblotting techniques. Whole laminin was not detected, whereas laminin fragments of 270, 155, 87, 56, and 14 kDa were detected at a mean total value of approximately 0.5 microgram/ml. The laminin fragments were assessed for their effect on cultured normal and ADPKD epithelial cells. Both cell types showed accelerated growth under these conditions. These findings suggest that basement membrane degradative fragments present in cyst fluid may contribute to cystic epithelial cell proliferation and may therefore be important in the pathogenesis of ADPKD.

Topics: Adult; Cell Division; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Exudates and Transudates; Female; Humans; Kidney Tubules; Laminin; Male; Middle Aged; Molecular Weight; Polycystic Kidney, Autosomal Dominant; Sodium Dodecyl Sulfate