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

Authors: Cai, Rong; Huang, Ming; Wang, Yinsheng

Published In Anal Chem, (2018 Dec 18)

Abstract: 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.

PubMed ID: 30433760 Exiting the NIEHS site

MeSH Terms: Cell Line, Tumor; Colonic Neoplasms/pathology; GTP-Binding Proteins/metabolism*; Guanosine Triphosphate/metabolism*; Humans; Isotope Labeling; Neoplasm Metastasis; Proteomics/methods*

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