Abstract 1268: Accurate measurement of serum total and free PSA using immunoaffinity depletion coupled to SRM: Correlation with clinical immunoassays
Authors: Liu Tao , Shi Tujin , Hossain Mahmud , Schepmoes Athena , Fillmore Thomas , Sokoll Lori J. , Chan Daniel , Leach Robin , Thompson Ian , Smith Richard D. , Kagan Jacob , Srivastava Sudhir , Rodland Karin D. , Camp David G. , Qian Wei-Jun 
Published: August 2012

Abstract
Accurate and reproducible quantification of proteins at the sub-ng/mL concentration range is required for the application of protein biomarkers to clinical assays using blood plasma or serum. The standard technique used in clinical labs for quantifying protein biomarkers for disease detection, monitoring and therapeutic intervention is sandwich immunoassay; although highly sensitive, the development of a specific immunoassay is time-consuming and associated with high costs due to the requirement for paired immunoaffinity reagents of high specificity. Recently, mass spectrometry-based methods, specifically, selected reaction monitoring mass spectrometry (SRM-MS), have been increasingly applied to measure low abundance biomarker candidates in tissues and biofluids, owing to high sensitivity and specificity, simplicity of assay configuration, and exceptional multiplexing capability. In this study, we report for the first time the development of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. With stable isotope dilution and external calibration, low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed high levels of correlation (R2 values ranging from 0.90 to 0.99) in independent clinical serum sample sets, including a set of 33 samples assayed in a blinded test. In addition, we developed an ultrasensitive antibody-free strategy for achieving accurate detection and quantification of proteins at ∼50 pg/mL level in human plasma/serum using high resolution reversed phase separations for target enrichment along with intelligent selection of target fractions via on-line SRM monitoring of internal standards and fraction multiplexing prior to SRM quantification. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring total and free PSA in human blood. Furthermore, the simultaneous measurement of the free and bound forms of PSA, as well as many other biomarkers that have similar complexation/binding characteristics, can be performed in a single multiplexed analysis using high-resolution liquid chromatographic separation coupled with SRM-MS. This work represents a major technological advance for achieving pg/mL levels of protein quantification in blood plasma without the need for specific antibodies for enrichment and holds great promise for broad applications in biomarker verification and systems biology applications.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1268. doi:1538-7445.AM2012-1268