Abstract 3872: Quantification of mutant SPOP proteins in prostate cancer using targeted proteomics

Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ∼10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for diagnosis, prognosis or targeted therapy of prostate cancer. To address this issue, selected reaction monitoring (SRM) and PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) -SRM mass spectrometry assays have been developed for quantifying mutant SPOP proteins. SRM assays for wild-type SPOP protein and 11 prostate cancer-derived mutations were developed. The presence of multiple lysine residues in the mutation regions precludes the use of conventional tryptic digestion. Arg-C was selected instead due to its superior performance in generating mutation site(s) containing SPOP peptides that are more suitable for SRM analysis comparing to other proteases (e.g., Asp-N). Although the resulting Arg-C peptides are longer and more hydrophobic than typical tryptic peptides, all the SRM assays showed a linear dynamic range of more than two orders of magnitude. The limits of quantification for the mutation site(s) containing peptides range from 10 to 100 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze 293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations, which agrees with RT-PCR results. It is unknown if this is related to activity of the SPOP protein. PRISM-SRM assays have shown further improvement in sensitivity. SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer. Citation Format: Hui Wang, Christopher Barbieri, Jintang He, Yuqian Gao, Chaochao Wu, Athena Schepmoes, Thomas Fillmore, Tujin Shi, Sung-Suk Chae, Dennis Huang, Juan Miguel Mosquera, Wei-Jun Qian, Richard Smith, Sudhir Srivastava, Jacob Kagan, David Camp, Karin Rodland, Mark Rubin, Tao Liu. Quantification of mutant SPOP proteins in prostate cancer using targeted proteomics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3872.