Journal: Molecular & Cellular Proteomics : MCP
Article Title: Systems-wide Analysis of K-Ras, Cdc42, and PAK4 Signaling by Quantitative Phosphoproteomics
Figure Lengend Snippet: Experimental workflow. A, K-Ras is a small GTPase that regulates the activity of a variety of downstream proteins including the Rho GTPase Cdc42. The PAK4 serine/threonine kinase is a direct effector of Cdc42 and regulates actin reorganization, microtubule stability, and cell polarity. B, To measure large-scale phosphorylation changes induced by constitutive K-Ras or Cdc42 signaling or PAK4 ablation, the quantitative label-free PTMscan® approach was employed (Cell Signaling Technology). Briefly, for each condition extracted proteins were digested with trypsin and separated from non-peptide material by solid-phase extraction with Sep-Pak C18 cartridges. Three phosphorylation motif antibodies were used serially to isolate phosphorylated peptides in independent immunoaffinity purifications (CDK substrate motif [K R]-pS-P-X-[K R], CK substrate motif pT-[D E]-X-[D E], PKD substrate motif l-X-R-X-X-p[S T]). The samples were run in duplicate and tandem mass spectra were collected with an LTQ-Orbitrap hybrid mass spectrometer. pLPC is an empty vector control.
Article Snippet: To measure quantitative changes in the levels of phosphorylation sites induced by perturbing K-Ras, Cdc42, or PAK4 signaling, we employed the label-free based PTMScan® method from Cell Signaling Technology ( 50 ) in wild-type and PAK4 knockout NIH3T3 cell lines expressing oncogenic K-Ras, activated Cdc42, or an empty vector control ( B ) (Materials and Methods).
Techniques: Activity Assay, Mass Spectrometry, Plasmid Preparation