Abstract Novel agents that inhibit targets in the hypoxia and MTOR pathways can achieve response rates of 30-60% in renal cell carcinoma (RCC); however, biomarkers have not yet been identified to measure degree of target inhibition and mechanism of biologic response in individual patients. We developed the use of a high-throughput, nanoscale immunoassay (NIA, Nanopro1000 Instrument, Protein Simple) to profile hypoxia pathways and downstream signaling in serially sampled clinical specimens from patients with RCC. NIA uses charge-based separation to distinguish multiple charged modifications of individual proteins, and measure relative ratios of individual unphosphorylated and phosphorylated isoforms. We first developed and optimized assays to measure therapeutic targets and proteins in hypoxia, proliferation and metabolic pathways (including CaIX, HIF, VEGFR, ERK, AKT, MYC, glutaminase) to analyze frozen surgical specimens, fine needle aspirates (FNA's) and blood PBMC specimens from patients with RCC. Next, we analyzed more than 200 FNA's from solid tumors, comparing RCC with paired adjacent non-tumor tissue and other epithelial malignancies. Basal MAPK signaling across the samples ranged across 3 logs for both normal and tumor tissue, demonstrating the difficulty in setting a threshold predictive of response based on a single pre-treatment measurement. To test whether analysis of serial specimens could detect changes, we prospectively collected and analyzed blood peripheral mononuclear cells from 20 RCC patients before and during treatment with standard targeted therapies including MTOR and tyrosine kinase inhibitors. We report for the first time that targeted inhibitors can preferentially inhibit or activate specific protein phospho-isoforms in patients with RCC. Our studies demonstrate that rapid and quantitative nanoproteomic profiling in very small amounts of serially sampled clinical specimen may accelerate translational studies for predictive proteomic biomarkers of response to targeted therapy. Citation Format: Alice C. Fan, John Leppert, Joanna E. Liliental, Liwen Xu, Thomas J. Metzner, Emelyn Shroff, Alia Yaghi, Christine Yost, James D. Brooks, Lauren C. Harshman, Chiara Sabatti, Sandhya Srinivas, Dean W. Felsher. Nano-scale proteomic profiles of response to targeted therapy in patients with RCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2283. doi:10.1158/1538-7445.AM2013-2283