Purification and identification of candidate biomarkers discovered using SELDI-TOF MS

Purification and identification of candidate biomarkers is a critical step in the biomarker development process, since it provides insight into the disease biology and facilitates the development of analyte-specific assays. Top-down biomarker discovery workflows like SELDI-TOF MS yield candidate markers that are identified based on native mass. Positive identification of these candidate biomarkers requires further enrichment and/or purification. While purification methods must be optimized for each protein target, there are two general workflows. Native peptides under approximately 4 kDa can be subjected to direct sequence analysis using a tandem mass spectrometer whereas proteins over approximately 4 kDa usually require proteolytic digestion prior to MS/MS analysis. In both cases, partial purification is usually necessary to enrich the candidate biomarker relative to other proteins in a complex biological mixture. This chapter provides detailed protocols for protein purification (including anion exchange, metal affinity, and reverse phase chromatography as well as SDS-PAGE) and identification (including protein processing, digestion, and database searching).