Abstract 2483: An antibody-independent, complementary SRM strategy for ultrasensitive and multiplexed quantification of cancer biomarker candidates

Abstract

Abstract Stable-isotope dilution coupled to targeted mass spectrometry (MS), applying selected reaction monitoring (SRM), provides good selectivity, reproducibility, sensitivity, and multiplexing capability rendering it highly suitable for robust, high-throughput and cost-effective verification of cancer biomarker candidates. The ability of SRM-MS to detect and quantify low abundance proteins present in blood plasma/serum at low ng/mL or even lower levels, however, relies on the front-end enrichment through specific affinity reagents (e.g., antibodies). To address this limitation, we have recently developed two antibody-independent, targeted quantification capabilities that enable protein quantification at low or sub ng/mL levels in plasma/serum. The first new capability, PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM, uses high resolution basic pH reversed-phase capillary liquid chromatographic (LC) separation with on-line SRM monitoring for the enrichment of target peptides and intelligent selection of target fractions for subsequent LC-SRM analysis that uses very short LC gradient to achieve better sensitivity and improved sample throughput. Application of PRISM-SRM enables highly sensitivity detection of several prostate cancer relevant proteins, e.g., prostate-specific antigen (PSA) and anterior gradient 2 (AGR2) proteins at ∼100 pg/mL level in serum, and distinct TMPRSS2-ERG fusion protein isoforms in cell line and tumor tissue samples. The front-end sample prefractionation often requires a relatively large amount of starting material (e.g., 25 ug in PRISM-SRM analysis) and increased instrument analysis time, and undesired sample losses are often encountered during multistep sample preparation. These can be effectively overcome by the second new capability, long-gradient separations coupled with SRM (LG-SRM). Our results show that LG-SRM enables reliable quantification of plasma proteins at low ng/mL levels in nondepleted human plasma/serum. Comparing to fractionation-based SRM methods, LG-SRM offers better sample throughput, requires minute amounts of starting materials (e.g., ∼4 μg), and provides at least 3-fold greater multiplexing capacity than conventional LC-SRM due to ∼3-fold increase in average peak widths. The complementary SRM strategy encompasses both PRISM-SRM and LG-SRM and is therefore highly attractive for the sensitive and multiplexed quantification of cancer biomarker candidates - PRISM-SRM offers ∼10X higher sensitivity, but LG-SRM requires much less sample and can be readily implemented. It is anticipated that these affinity reagent-free, highly sensitive proteomics measurements made possible by this complementary SRM strategy will bring new biological insights and perspectives to prostate cancer research as well as other cancers. Citation Format: Tujin Shi, Jintang He, Chaochao Wu, Thomas L. Fillmore, Athena A. Schepmoes, Mark Rubin, Javed Siddiqui, John Wei, Arul Chinnaiyan, Alvin Liu, Richard D. Smith, Jacob Kagan, Sudhir Srivastava, Karin D. Rodland, Wei-Jun Qian, Tao Liu, David G. Camp. An antibody-independent, complementary SRM strategy for ultrasensitive and multiplexed quantification of cancer biomarker candidates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2483. doi:10.1158/1538-7445.AM2014-2483