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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.

Radiotherapy constitutes a major therapeutic modality for early management of breast cancer. Despite the high efficacy in treating breast cancer (BC), radiation resistance and tumor recurrence are major hurdles in breast cancer radiotherapy. Herein, stable isotope labeling by amino acids in cell culture (SILAC) was employed, along with the parallel-reaction monitoring (PRM)-based targeted quantitative proteomic method, to examine the differences in kinase protein expression in MCF-7 and MDA-MB-231 breast cancer cells and their corresponding radioresistant C6 and C5 clones. We quantified the relative protein expression levels of 300 and 281 kinases in C5/MDA-MB-231 and C6/MCF-7 pairs of breast cancer cells, respectively. We also showed that TAF9, which was one of the differentially expressed kinases, enhances radiation resistance in breast cancer cells. Moreover, a correlation analysis of gene expression suggested TAF9s role in upregulating the expression of genes involved with radioresistance. Overall, our study uncovered a large number of differentially expressed kinases accompanied with the acquisition of radioresistance and revealed a role of TAF9 in promoting radioresistance in breast cancer.

Protein α-N-methylation is an evolutionarily conserved type of post-translational modification; however, little is known about the regulatory mechanisms for this modification. Methylation at the N6 position of adenosine in mRNAs is dynamic and modulates their stability, splicing, and translational efficiency. Here, we found that the expression of N-terminal methyltransferase 1 (NTMT1) protein is altered by depletion of those genes encoding the reader/writer/eraser proteins of N6-methyladenosine (m6A). We also observed that MRG15 is N-terminally methylated by NTMT1, and this methylation could also be modulated by reader/writer/eraser proteins of m6A. Together, these results revealed a novel m6A-based epitranscriptomic mechanism in regulating protein N-terminal methylation.