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In this chapter we describe how mass spectrometry-based quantitative protein measurements by multiple reaction monitoring (MRM) have opened up the opportunity for the assembly of large panels of candidate protein biomarkers that can be simultaneously validated in large clinical cohorts to identify diagnostic protein biomarker signatures. We outline a workflow in which candidate protein biomarker panels are initially assembled from multiple diverse sources of discovery data, including proteomics and transcriptomics experiments, as well as from candidates found in the literature. Subsequently, the individual candidates in these large panels may be prioritised by application of a range of bioinformatics tools to generate a refined panel for which MRM assays may be developed. We describe a process for MRM assay design and implementation, and illustrate how the data generated from these multiplexed MRM measurements of prioritised candidates may be subjected to a range of statistical tools to create robust biomarker signatures for further clinical validation in large patient sample cohorts. Through this overall approach MRM has the potential to not only support individual biomarker validation but also facilitate the development of clinically useful protein biomarker signatures.

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