The proteome refers to the collection of proteins in a given biological organism or system under a particular set of environmental conditions. The study of proteins, referred to as proteomics, is performed to identify the components of a particular proteome and analyze global changes in protein expression in response to different stimuli. This leads to an understanding of physiological and pathological states of an organism through a comprehensive analysis of biological processes. From a protein complex to whole cell, proteome analysis deals with highly complex mixtures, requiring more than one analytical dimension to achieve the high resolving power necessary for reliable analysis. In 1975, O’Farrell and Klose described two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)1,2 that could resolve complex protein mixtures into thousands of spots. Years later, upon the development of matrix-assisted laser desorption ionization (MALDI)3 and electrospray ionization (ESI),4,5 combined with database searching,6-9 the field of proteomics began to grow dramatically. Researchers were able to characterize complex mixtures of proteins and gain novel biological insights. Despite the longstanding success of 2D-PAGE coupled with mass spectrometry, several fundamental issues with the technology, including the challenges of identifying low-abundance proteins,10-12 membrane proteins,13 and proteins with extremes in isoelectric point (pI) and molecular weight (MW),14,15 drove researchers to develop alternative approaches for the separation of complex mixtures. This led to the emergence of shotgun proteomics based on the coupling of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Similar to the shotgun genomic sequencing approach in which DNA is broken into smaller pieces prior to sequencing and reassembled in silico, proteins are first digested into peptides and then analyzed by multidimensional chromatography coupled to tandem mass spectrometry (MS/MS). Thousands of tandem mass spectra are then compared to theoretical tandem mass spectra using database searching algorithms for the identification of proteins in the sample (Figure 1b). The inability of one-dimensional (1D) separation techniques to resolve complex biological samples for shotgun proteomics has required the development of multidimensional separation methods. A multidimensional separation includes two or more independent separation techniques (i.e., ion exchange, size exclusion, reversed phase, and affinity) coupled together for the analysis of a single sample. * Address correspondence to this author at the Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110 [telephone (816) 9264457; fax (816) 926-4694; e-mail mpw@stowers-institute.org]. † These authors contributed equally to this work. 3654 Chem. Rev. 2007, 107, 3654−3686