Targeted Quantitative Proteomic Approach for Probing Altered Protein Expression of Small GTPases Associated with...

Genes encoding the small GTPases of the Ras superfamily are among the most frequently mutated or dysregulated in human cancer. No systematic studies, however, have yet been conducted for assessing the implications of small GTPases in the metastatic transformation of colorectal cancer (CRC). By utilizing a recently established high-throughput multiple-reaction monitoring (MRM)-based workflow together with stable isotope labeling by amino acids in cell culture (SILAC), we investigated comprehensively the relative expression of the small GTPase proteome in a pair of matched primary/metastatic CRC cell lines (SW480/SW620). Among the 83 quantified small GTPases, 25 exhibited at least a 1.5-fold difference in protein expression in SW480 and SW620 cells. In particular, SAR1B protein was found to be substantially down-regulated in SW620 relative to SW480 cells. In addition, bioinformatic analyses revealed that diminished SAR1B mRNA expression is significantly associated with higher CRC stages and unfavorable patient prognosis, in support of a potential role of SAR1B in suppressing CRC metastasis. In addition, diminished SAR1B expression could stimulate epithelial-mesenchymal transition (EMT), thereby promoting motility and in vitro metastasis of SW480 cells. In summary, we profiled systematically, by employing an MRM-based targeted proteomic method, the differential expression of small GTPase proteins in a matched pair of primary/metastatic CRC cell lines. Our results revealed the potential roles of SAR1B in suppressing CRC metastasis and in the prognosis of CRC patients.