Figure 1.
Analysis of WT and β3–/– embryonic fibroblasts for quantitative proteomics. A: β3 Integrin deficiency does not change morphology but alters immunofluorescence pattern of paxillin (left panels). Phase-contrast images of WT (top) and β3–/– (bottom) fibroblasts under ×20 magnification (right panels). Merged immunofluorescence images of WT (top) and β3–/– (bottom) fibroblasts stained with anti-paxillin-FITC which localizes to focal contacts (white arrows), phalloidin-Alexa594 (actin), and DAPI (chromatin). Scale bar (bottom right) represents 10 μm. B: Outline of quantitative experimental procedure. Mouse embryonic fibroblasts were seeded onto fibronectin-coated plates, harvested and sub-fractionated. One hundred micrograms of cytosolic protein from WT and β3–/– cells were utilized for ICAT labeling and 1D-PAGE. After Coomassie staining, eight equal-sized gel slices (corresponding to mass ranges of <15, 15-25, 25-35, 35-50, 50-65, 65-100, 100-150, and >150 kDa) were excised and trypsin digested. Digested peptides were further purified over an avidin affinity cartridge. Peptides were eluted from the cartridge then acid-cleaved to release the ICAT-labeled peptides from the linker and subjected to nanoLC-MS/MS analysis. Details of the multidimensional procedure and the nanoLC-MS/MS are described in the Materials and Methods.