Generation and analysis of multidimensional protein identification technology datasets.

Systems that couple two dimensional liquid chromatography (LC/LC) with tandem mass spectrometry are widely used in modern proteomics. One such system, multidimensional protein identification technology (MudPIT), couples strong cation exchange chromatography and reversed phase chromatography to tandem mass spectrometry in a single microcapillary column. Using database searching algorithms like SEQUEST and additional computational tools, researchers are able to analyze in great detail complex peptide mixtures generated from biofluids, tissues, cells, organelles, or protein complexes. This chapter describes the use of MudPIT on modern mass spectrometry instrumentation and describes a data analysis pipeline designed to provide low false positive rates and quantitative datasets.