To analyze highly complex mixtures of proteins, a combination of 2D-PAGE and Mass Spectrometry (MS) is usually used. However, this can be problematic when dealing with low abundance proteins, membrane-associated or bound proteins, or proteins with extremes in pI or molecular mass. Alternative methods instead couple Multidimensional Chromatography with MS. MuDPIT, or “Multidimensional Protein Identification Technology” couples 2D-Liquid Chromatography with with MS/MS to resolve proteins from complex mixtures. David M. Schieltz and Michael P. Washburn have described a method for proteomic analysis using MuDPIT, which is available freely as one of our featured protocols.