MudPIT: A powerful proteomics tool for discovery.

Extract: Biochemists have broken barriers in the capabilities of proteomics, providing cell biologists and medical researchers unparalleled opportunities to analyze complex protein mixtures. The next task is to generate new ideas in the choice and preparation of materials to analyze. Traditional proteomics methodologies separate complex protein samples by isoelectric point and molecular weight using 2-dimensional gels. Patterns can be compared between samples, but to determine which protein is changing requires isolating individual protein spots, proteolyzing these, and analyzing the mass of each peptide by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The measured peptide masses are searched against the predicted mass values for theoretical digestion of proteins in a sequence database, and the protein is identified by a statistically significant number of matches. Multidimensional Protein Identification Technology (MudPIT) eliminates gel separations. Instead, biochemical fractions containing many proteins are directly proteolyzed and the enormous number of peptides generated, are separated by 2-dimensional liquid chromatography before entering the mass spectrometer. Instead of MALDI-TOF, the procedure employs tandem mass spectrometry so that, after the mass of a peptide is measured, the peptide is fragmented using a collision-induced dissociation cell and the masses of the fragmentation products are determined.