The order of steps in the workflow and the detailed experimental protocol can be sources of variability. a holoenzyme complex, and specific protein connection partners may be present in nonstoichiometric amounts. For example, catalytic subunits such as protein phosphatase 1 (PP1) can interact with a spectrum of option protein partners, which therefore bind nonstoichiometrically to generate a range of holoenzymes with different specificities (for review seeMoorhead et al., 2007). This can make it hard to distinguish specific but low large quantity interacting proteins from the larger quantity of low affinity, but abundant, contaminant proteins that are inevitably recovered using popular methods such as pull-down or immunoprecipitation strategies. A key PFI-3 goal in most areas of cell biology, consequently, is the characterization of the protein components of multiprotein complexes through the reliable identification of specific protein interaction partners. Any putative connection partner recognized either through PFI-3 affinity purification or biochemical fractionation must be validated to confirm its physiological relevance. These downstream validation experiments, involving detailed molecular characterization, are both expensive and time consuming and thus it is imperative to focus resources on those subsets of potential relationships with a high probability of biological significance. Continuing improvement in the level of sensitivity and resolution of the mass spectrometric technology for protein recognition, such as, allows for the recognition of ever larger numbers of proteins in immunoaffinity and pull-down experiments. In addition to bona fide interaction partners, however, these expanding lists include improved numbers of contaminant proteins, including those that bind nonspecifically to the affinity matrix. The problem of nonspecific binding PFI-3 cannot be conquer satisfactorily using high stringency PP2Abeta purification methods; although this can reduce the level of nonspecific binding, it will inevitably also remove low large quantity and low affinity specific partner proteins. The most effective strategy must consequently preserve all specific connection events, which inevitably results in a large number of nonspecific proteins also copurifying that must be recognized and discarded. To solve this problem, we as well as others have demonstrated that a quantitative mass spectrometrybased approach combined with isotope labeling can help to distinguish which of the many proteins identified inside a pull-down or immunoprecipitation experiment represent specific binding. This is done from the inclusion of a negative control, which provides a background of contaminant proteins that bind nonspecifically to the affinity matrix and/or the fusion tag, against which proteins that bind specifically to the protein of interest clearly stand out (for review seeVermeulen et al., 2008). For example, using a combination of stable isotope labeling with amino acids in cell tradition (SILAC)centered quantitative proteomics (Ong et al., 2002) with immunoprecipitation of GFP-tagged fusion proteins, we revealed variations in binding partners for two different isoforms of the nuclear protein phosphatase, PP1 (Trinkle-Mulcahy et al., 2006). Additional groups have used a similar approach based on tagged bait proteins to map the spectrum PFI-3 of human being 26S proteasome interacting proteins (Wang and Huang, 2008) and to detect dynamic users of transcription element complexes (Mousson et al., 2008). Isotope-based quantitative methods have also been used to define tagged protein complexes in candida (Ranish et al., 2003;Tackett et al., 2005) and both tagged and endogenous protein complexes in mammalian cells (Blagoev et al., 2003;Cristea et al., 2005;Selbach and.