期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 109, 期 38, 页码 15395-15400出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1204366109
关键词
biomarker verification; systems biology; targeted proteomics
资金
- National Institutes of Health (NIH) [DP2OD006668]
- NCI Early Detection Research Network [Y01-CN-05013-29]
- NIH [8P41 GM103493, 5P41 RR018522, CA111244, U24-CA-160019]
- US Department of Energy (DOE)
- DOE/Biological and Environmental Research (BER)
- DOE [DE-AC05-76RL0 1830]
Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)-based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano-liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50-100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.
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