Phosphoproteome analysis of trastuzumab resistant breast cancer cells by electron transfer dissociation mass spectrometry

LB-152

Targeted therapy represents a novel regimen of caner management, which is exemplified by a new generation of anti-kinase drugs. These drugs specifically inhibit a kinase or multiple kinases that play critical roles in cancer development and advance. While the advantages of targeted therapy are obvious, such therapy often relapses and results in drug resistance for advanced cancers. Unraveling anti-kinase drug resistance mechanisms would facilitate novel drug development and in turn better understanding of drug cytotoxicity. The MS-based phosphoproteomic technologies provide novel approaches to the identification of phosphosignatures unique to a specific type or subtype of cancer and drug responsive biomarkers for personalized therapy. In this study, the phosphoproteome of trastuzumab resistant human breast cancer JIMT-1 cells was analyzed using improved titanium dioxide (TiO₂) phosphopeptide enrichment and electron transfer dissociation (ETD) and collision-induced dissociation (CID) mass spectrometry. High specific phosphopeptide enrichment was achieved using TiO₂ columns in combination with improved peptide methylation technique, which resulted in identification of hundreds of phosphoproteins from one-dimensional LC-MS/MS analysis. Many phosphoproteins related to breast cancer development, such as rhabdomyosarcoma antigen MU-RMS-40.12, Src substrate cortactin and epidermal growth factor receptor substrate 15-like 1 (Eps15L1 or Eps15R), were detected. Particularly, the protein Eps15R is a tyrosine kinase substrat that has the ability to bind to the clathrin adaptor protein complex adaptor protein 2. Studies have demonstrated that Eps15R is an essential component of the endocytic machinery and play important roles in EGFR ligand internalization. Although more studies should be conducted to investigate whether such proteins are involved in trastuzumab resistance of this type of breast cancer cells, the present phosphoproteome analysis provide valuable information and novel insights into the mechanisms of anticancer drug resistance. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.