Targeted Quantitative Profiling of GTP-Binding Proteins in Cancer Cells Using Isotope-Coded GTP Probes

GTP-binding proteins play important roles in many essential biological processes, including cell signaling, trafficking, and protein synthesis. To assess quantitatively these proteins at the whole proteome level, we developed a high-throughput targeted proteomic method based on the use of isotope-coded GTP probes and multiple-reaction monitoring (MRM) analysis. Targeted proteins were labeled with desthiobiotin-GTP probes, digested with trypsin, and the ensuing desthiobiotin-conjugated peptides were enriched with streptavidin beads for LC-MS/MS analysis. We also established a Skyline MRM library based on shotgun proteomic data acquired for 12 different human cell lines. The library contained 605 tryptic peptides derived from 217 GTP-binding proteins, representing approximately 60% of the annotated human GTP-binding proteome. By using this library, in conjunction with isotope-coded GTP probes and scheduled LC-MRM analysis, we investigated the differential expression of GTP-binding proteins in a pair of primary/metastatic colon cancer cell lines (SW480 and SW620). We were able to quantify 97 GTP-binding proteins, and we further validated the differential expression of several GTP-binding proteins by Western blot analysis. Together, we developed a facile targeted quantitative proteomic method for the high-throughput analysis of GTP-binding proteins and applied the method for probing the altered expression of these proteins involved in colon cancer metastasis.