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Phosphoproteomics Approaches

Profiling signaling polarity in chemotactic cells.

Serum starved Cos-7 cells were induced to extend pseudopodia (PD) through 3.0-m pores of fibronectin coated filters with LPA gradient (1-3). The PD and cell bodies (CB) were harvested in a urea buffer that is compatible for mass spectrometry analysis. For the identification of the total PD and CB proteome, the lysate from each fraction was digested with trypsin and the resulting peptides were directly identified by LC-MS/MS, the relative abundance of each protein in PD and CB was estimated by calculating the ratio of the peptide counts associated with the protein (PD/CB) (Table 1). The proteins from major cellular organelles were grouped based on the terms of GeneOntology (Table 2). For the quantitative identification of phosphosites in PD and CB, we digested the lysate from PD and CB with trypsin, and labeled the resulting peptides with 18O and 16O, respectively, using the trypsin catalyzed dual step 18O/16O labeling approach. Phosphopeptides from the labeled peptides were further enriched with immobilized metal affinity chromatography (IMAC) and quantitatively identified using LC-MS/MS or MS3 (Table 3), and the conservative analysis for each phosphosite across species was performed using the Blastpro program (4).

  1. Wang, Y. & Klemke, R. L. (2006) in Methods in Molecular Biology, ed. Coutts, A. S. (Humana Press Inc., Totowa, NJ), Vol. 370, pp. 55-66. PubMed
  2. Wang, Y., Ding, S. J., Wang, W., Jacobs, J. M., Qian, W. J., Moore, R. J., Yang, F., Camp, D. G., Smith, R. D. & Klemke, R. L. (2007) Profiling signaling polarity in chemotactic cells. Proc Natl Acad Sci U S A 104: 8328 - 33. PubMed
  3. Cho, S. Y. & Klemke, R. L. (2002) Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold. J Cell Biol 156: 725-36. PubMed
  4. Wang, Y., Hanley, R. & Klemke, R. L. (2006) Computational methods for comparison of large genomic and proteomic datasets reveal protein markers of metastatic cancer. J Proteome Res 5: 907-15. PubMed

From: Wang et al. (2007) "Profiling signaling polarity in chemotactic cells." Proc. Natl. Acad Sci. US 104:8328-8333.

Supplementary Table 1. Identification and Quantitation of Proteins from Cell Body (CB) and Pseudopodium (PD) fractions            Published version (Excel spreadsheet)

Supplementary Table 2. Proteins in major organelles and subcellular compartments            Published version (Excel spreadsheet)

Supplementary Table 3. Quantitation of phosphopeptides identified in Pseudopodium (PD) and Cell Body (CB) fractions            Published version (Excel spreadsheet)