Development of tamoxifen resistance remains a tremendous challenge for the treatment of estrogen-receptor (ER)-positive breast cancer. Small GTPases of the Ras superfamily play crucial roles in intracellular trafficking and cell signaling, and aberrant small-GTPase signaling is implicated in many types of cancer. In this study, we employed a targeted, quantitative proteomic approach that relies on stable-isotope labeling by amino acids in cell culture (SILAC), gel fractionation, and scheduled multiple-reaction-monitoring (MRM) analysis, to assess the differential expression of small GTPases in MCF-7 and the paired tamoxifen-resistant breast cancer cells. The method displayed superior sensitivity and reproducibility over the shotgun-proteomic approach, and it facilitated the quantification of 96 small GTPases. Among them, 13 and 10 proteins were significantly down- and up-regulated (with >1.5-fold change), respectively, in the tamoxifen-resistant line relative to in the parental line. In particular, we observed a significant down-regulation of RAB31 in tamoxifen-resistant cells, which, in combination with bioinformatic analysis and downstream validation experiments, supported a role for RAB31 in tamoxifen resistance in ER-positive breast-cancer cells. Together, our results demonstrated that the targeted proteomic method constituted a powerful approach for revealing the role of small GTPases in therapeutic resistance.
