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.