Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells

Abstract

Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress.

High-coverage proteome analysis reveals the first insight of protein modification systems in the pathogenic spirochete Leptospira interrogans

“…After visualizing on LAS-4000 (Fujifilm), the gel was stained by Coomassie brilliant blue. The gel bands containing LipL32 were excised and were in-gel digested by trypsin, as described previously [49].…”

Quantitative Phosphoproteome Profiling of Wnt3a-mediated Signaling Network

“…The two gel lanes ("0 min-1 min," the combined cell samples stimulated with Wnt3a for 0 or 1 min; "0 min-30 min," the combined cell samples stimulated with Wnt3a for 0 or 30 min) were cut into 26 slices, respectively. The excised slices were subjected to in-gel trypsin digestion as described before (26).…”

“…Eluted phosphoproteins were dialyzed, lyophilized, and reconstituted by an equal volume (250 l) of 4ϫ sample buffer (40% glycerol, 8% SDS, 250 mM Tris-HCl, 4% ␤-mercaptoethanol). To validate the efficacy of phosphoprotein enrichment in the elution fraction, 10 g of proteins of the total cell lysate, elution fraction, and flow-through fraction from the phosphoprotein purification kit were separated by 12.5% SDS-PAGE followed by silver staining (26) or Western blotting analysis using mixed phosphoserine, phosphothreonine, and phosphotyrosine monoclonal antibodies. For Western blotting verification of SILAC results, the eluted phosphoprotein fractions were loaded at the same volume of 10 l. After transfer, the nitrocellulose membranes were blocked by 1ϫ Net-gelatin (150 mM NaCl, 5 mM EDTA, 50 mM TrisHCl, pH 7.5, 0.05% Triton X-100, 0.25% gelatin) and then incubated with the corresponding primary antibodies followed by horseradish peroxidase-conjugated or IRDye 800CW-conjugated affinity-purified secondary antibodies.…”

Quantitative Proteomics Analysis of Inborn Errors of Cholesterol Synthesis

“…Gel Staining-After 2-DE, the analytic gels were stained with ammoniacal silver nitrate as described previously (24). Briefly, the analytic gels were washed for 5 min in water and soaked for 1 h in ethanol:acetic acid:water (40:10:50) followed by overnight immersion in ethanol:acetic acid:water (5:5:90).…”

“…Two-dimensional Electrophoresis-2-DE was performed as described previously (24). Briefly, IPG-IEF was performed on an Ettan IPGphor 3 IEF System (GE Healthcare).…”