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There are currently no widely accepted biomarkers for non-invasive diagnosis or screening of bladder cancer. There is, therefore, a compelling need to develop more reliable bladder cancer biomarkers, particularly those which can be measured in body fluids. In this book chapter, we describe the proteomic workflow which we used to develop a non-invasive assay for the detection of human bladder tumor in urine specimens. A six-protein biomarker panel was generated by a combination of untargeted mass-spectrometry-based biomarker discovery using an “isobaric tags for relative and absolute quantitation” (iTRAQ) platform, and subsequent biomarker verification using a targeted multiple-reaction-monitoring mass spectrometry (MRM-MS) approach.

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