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Proteins are the ultimate effectors of essentially all cellular processes.Thus, in contrast to most other types of biomolecule, their levels and post-translational modifications, directly reflect the activities of cells and tissues.Given this background, progress to date in identifying proteins or peptides whose abundances correlate with a particular pathological condition has been disappointing.In part this is due to well-known challenges associated with linking biomarker discovery with well-established methods for validation.However, it is also clear that much remains to be done in the discovery phase to yield stronger candidate biomarkers.This talk will focus on improvements in experimental study design that more readily identify proteins whose levels reflect a disease state.In particular, it will emphasize the importance of using truly independent sets of samples to confirm the differential abundance of candidate biomarkers, rather than the split-sample approach most commonly used today.The talk will be illustrated by recent mass spectrometry-based studies to identify candidate biomarkers for bovine tuberculosis infection and also stage-specific markers of chronic obstructive pulmonary disease (COPD).