Multi-Site Assessment of the Precision and Reproducibility of Multiple Reaction Monitoring-Based Measurements of Proteins in Plasma

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
Nature Biotechnology (Impact Factor: 41.51). 07/2009; 27(7):633-41. DOI: 10.1038/nbt.1546
Source: PubMed


Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low mug/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.

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    • "The use of standards in SRM approaches is critical for the quantitative performance of the assay and the reproducibility both within lab and between labs [77] [78]. Most often ADME proteomics have used stable isotope labeled (SIL) peptides, but synthetic´concatamers´(QconCAT) [71] and whole proteins stable isotope labeled by amino acids in cell culture (SILAC) [79] [80], have also been employed. "
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    • "Our findings are in-line with a recent multi-laboratory study undertaken to assess reproducibility, dynamic range, limit of detection and quantification issues from MRM/SRM based analysis using a standardized protocol. The authors found that the results are highly reproducible across laboratories and instrument platforms [116] "
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    • "A suitable set of transitions with accurate precursor ion retention times constitutes a definitive assay for that protein (Lange et al., 2008). Once a set of transitions is established, the assay can be multiplexed with great reproducibility, even across laboratories (Addona et al., 2009). MRM coupled with subcellular fractionation can provide a comprehensive analysis of protein quantitation with wide dynamic range in human autopsy brain. "
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