Tujin Shi (Presenter)
Biological Sciences Division
Bio: N/A
Authorship: Tujin Shi1, Hui Wang1, Ehwang Song1, Song Nie1, Thomas L. Fillmore2, Athena A. Schepmoes1, Mark A. Rubin3, Alvin Y. Liu4, Richard D. Smith1, Jacob Kagan5, Sudhir Srivastava5, Wei-Jun Qian1, Tao Liu1, and Karin D. Rodland1
1Biological Sciences Division and 2 EMSL, Pacific Northwest National Laboratory, 3Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, 4Department of Urology, University of Washington, 5Division of Cancer Prevention, National Cancer Institute
| Short Abstract Selected reaction monitoring (SRM)-based targeted quantification 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 (i.e., PRISM-SRM and LG-SRM) that enable protein quantification at low or sub ng/mL levels in plasma/serum. Relevant applications for protein biomarker quantification will be presented. |
Long Abstract
Introduction:
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.
Methods and Results
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 ¥ìg), and provides at least 3-fold greater multiplexing capacity than conventional LC-SRM due to ¡3-fold increase in average peak widths.
Conclusions:
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.
References & Acknowledgements:
Parts of this work were supported by a National Cancer Institute Early Detection Research Network Interagency Agreement (No. Y01-CN-05013-29)and National Institutes of Health grants U24-CA-160019, P41GM103493, DP2OD006668, UC4DK104167.
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IP Royalty: no
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