title: Purification and identification of candidate biomarkers discovered using SELDI-TOF MS.
abstract: 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).
authors.affiliation: Bio-Rad Laboratories, Malvern, PA, USA. abulman@msn.com
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Bulman
authors.forename: Enrique A
authors.initials: EA
authors.lastname: Dalmasso
mesh_terms: D000478:Alkylation; D015415:Biomarkers / Q000032:analysis / Q000097:blood / Q000134:cerebrospinal fluid / Q000652:urine; D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods*; D057225:Data Mining; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D059748:Proteolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D015415:Biomarkers; D010446:Peptide Fragments