Targeted Quantitative Profiling of GTP-Binding Proteins Associated with Metastasis of Melanoma Cells

Metastasis
is a major obstacle in the therapeutic intervention
of melanoma, and several GTP-binding proteins were found to play important
roles in regulating cancer metastasis. To assess systematically the
regulatory roles of these proteins in melanoma metastasis, we employed
a targeted chemoproteomic method, which relies on the application
of stable isotope-labeled desthiobiotin-GTP acyl phosphate probes
in conjunction with scheduled multiple-reaction monitoring (MRM),
for profiling quantitatively the GTP-binding proteins. Following probe
labeling, tryptic digestion, and affinity pull-down of desthiobiotin-conjugated
peptides, differences in expression levels of GTP-binding proteins
in two matched pairs of primary/metastatic melanoma cell lines were
measured using liquid chromatography–MRM analysis. We also
showed that among the top upregulated proteins in metastatic melanoma
cells, AK4 promotes the migration and invasion of melanoma cells;
overexpression of AK4 in primary melanoma cells leads to augmented
migration and invasion, and reciprocally, knockdown of AK4 in metastatic
melanoma cells results in repressed invasiveness. In summary, we examined
the relative expression levels of GTP-binding proteins in two pairs
of primary/metastatic melanoma cell lines. Our results confirmed some
previously reported regulators of melanoma metastasis and revealed
a potential role of AK4 in promoting melanoma metastasis.