A simple and highly reproducible method for discovering potential disease markers in low abundance serum proteins

SUMMARY Serum provides a link between many human organs, tissues, and cells, and is one of the most informative body fluids. However, the presence of 22 abundant proteins and a large dynamic range of numerous other proteins make quantitative analysis of low abundance proteins challenging. Here, we describe simple and easy to use pretreatment techniques for serum combined with high abundant protein removal and reverse-phase high-performance liquid chromatography (RP-HPLC) separation, each step of which was optimized to minimize the loss of proteins and increase reproducibility. This method can be used to discover disease-specific biomarker proteins in concentrations of the ng/mL range, and furthermore, be used as a base strategy to comparatively analyze the deep proteome in the pg/mL range.