The discovery of candidate biomarkers within the entire proteome is one of the most important and challenging goals in proteomic research. Mass spectrometry-based proteomics is a modern and promising technology for semiquantitative and qualitative assessment of proteins, enabling protein sequencing and identification with exquisite accuracy and sensitivity. For mass spectrometry analysis, protein extractions from tissues or body fluids and subsequent protein fractionation represent an important and unavoidable step in the workflow for biomarker discovery. Following extraction of proteins, the protein mixture must be digested, reduced, alkylated, and cleaned up prior to mass spectrometry. The aim of our chapter is to provide comprehensible and practical lab procedures for sample digestion, protein fractionation, and subsequent mass spectrometry analysis.
Keywords: Biomarker discovery; In-gel digestion of proteins; In-solution digestion of proteins; LC-MS/MS; LTQ Orbitrap; Mass spectrometry; Proteomics; SEQUEST; Silver stain.