Analytical platform evaluation for quantification of ERG in prostate cancer using protein and mRNA detection methods

作者：Jintang He, Athena Schepmoes, Tujin Shi, Chaochao Wu, Thomas Fillmore, Yuqian Gao, Richard Smith, Weijun Qian, Karin Rodland, Tao Liu, David Camp, Anshu Rastogi, Shyh‐Han Tan, Wusheng Yan, Ahmed A. Mohamed, Wei Huang, Sreedatta Banerjee, Jacob Kagan, Sudhir Srivastava, David G. McLeod, Shiv Srivastava, György Petrovics, Albert Dobi, Alagarsamy Srinivasan · 发表于：Journal of Translational Medicine · 年份：2015 · DOI：10.1186/s12967-015-0418-z · 被引用次数：28 · 研究领域：Prostate Cancer Treatment and Research、Advanced Proteomics Techniques and Applications、Prostate Cancer Diagnosis and Treatment

BACKGROUND: The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer. METHODS: An antibody-free assay for ERG3 protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERG negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment). Enzyme-linked immunosorbent assay (ELISA), western blot, NanoString, and qRT-PCR were also used in the analysis of these samples. RESULTS: Recombinant ERG3 protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine. The sensitivity of the in-house ELISA was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR ex...