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Shi, Tujin; Fillmore, Thomas L; Sun, Xuefei; Zhao, Rui; Schepmoes, Athena A; Hossain, Mahmud; Xie, Fang; Wu, Si; Kim, Jong-Seo; Jones, Nathan; Moore, Ronald J; Paša-Tolić, Ljiljana; Kagan, Jacob; Rodland, Karin D; Liu, Tao; Tang, Keqi; Camp, David G; Smith, Richard D; Qian, Wei-Jun
Proceedings of the National Academy of Sciences - PNAS, 09/2012, Letnik: 109, Številka: 38Journal Article
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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