␤-arrestins are cytosolic proteins that form complexes with seventransmembrane receptors after agonist stimulation and phosphorylation by the G protein-coupled receptor kinases. They play an essential role in receptor desensitization and endocytosis, and they also serve as receptor-regulated signaling scaffolds and adaptors. Moreover, in the past decade, a growing list of protein-protein interactions of ␤-arrestins pertinent to these functions has been documented. The discovery of several novel functions of ␤-arrestins stimulated us to perform a global proteomics analysis of ␤-arrestininteracting proteins (interactome) as modulated by a model seventransmembrane receptor, the angiotensin II type 1a receptor, in an attempt to assess the full range of functions of these versatile molecules. As determined by LC tandem MS, 71 proteins interacted with ␤-arrestin 1, 164 interacted with ␤-arrestin 2, and 102 interacted with both ␤-arrestins. Some proteins bound only after agonist stimulation, whereas others dissociated. Bioinformatics analysis of the data indicates that proteins involved in cellular signaling, organization, and nucleic acid binding are the most highly represented in the ␤-arrestin interactome. Surprisingly, both S-arrestin (visual arrestin) and X-arrestin (cone arrestin) were also found in heteromeric complex with ␤-arrestins. The ␤-arrestin interactors distribute not only in the cytoplasm, but also in the nucleus as well as other subcellular compartments. The binding of 16 randomly selected newly identified ␤-arrestin partners was validated by coimmunoprecipitation assays in HEK293 cells. This study provides a comprehensive analysis of proteins that bind ␤-arrestin isoforms and underscores their potentially broad regulatory roles in mammalian cellular physiology. mass spectrometry ͉ seven-transmembrane receptor ͉ angiotensin II type 1a receptor ͉ interactome ͉ signal transduction S even-transmembrane receptors (7TMRs), the largest group of plasma membrane receptors, classically signal via activation of heterotrimeric G proteins and generation of second messengers such as cAMP, DAG, and IP3. Their signaling is rapidly quenched by a universal mechanism involving two families of proteins. G protein-coupled receptor kinases phosphorylate activated receptors, thereby promoting the binding of ␤-arrestin molecules (␤-arrestin 1 or 2, aka arrestin 2 and 3) or in the case of rhodopsin, visual arrestin (aka arrestin 1). The arrestin molecules ''desensitize'' the receptors by sterically inhibiting further G protein activation (1, 2).Over the past decade, however, a variety of additional functions of ␤-arrestins have been discovered. These include important roles in clathrin-mediated endocytosis of receptors and as signal transducers for a growing list of effector pathways such as MAP kinases, AKT, and phosphatidylinositol 3-kinase (PI3-kinase). Both the endocytic and signaling roles of ␤-arrestins rely on their ability to serve as adaptors and scaffolds that engage in regulated interactions with a variety of cell...

We previously reported the metabolic 15 N labeling of a rat where enrichment ranged from 94% to 74%. We report here an improved labeling strategy which generates 94% 15 N enrichment throughout all tissues of the rat. A high 15 N enrichment of the internal standard is necessary for accurate quantitation, and thus, this approach will allow quantitative mass spectrometry analysis of animal models of disease targeting any tissue.

Many neurological disorders are caused by perturbations during brain development, but these perturbations cannot be readily identified until there is comprehensive description of the development process. In this study, we performed mass spectrometry analysis of the synaptosomal and mitochondrial fractions from three rat brain regions at four postnatal time points. To quantitate our analysis, we employed 15 N labeled rat brains using a technique called SILAM (Stable Isotope Labeling in Mammals). We quantified 167,429 peptides and identified over 5000 statistically significant changes during development including known disease associated proteins. Global analysis revealed distinct trends between the synaptic and non-synaptic mitochondrial proteomes and common protein networks between regions each consisting of a unique array of expression patterns. Finally, we identified novel regulators of neurodevelopment that possess the identical temporal pattern of known regulators of neurodevelopment. Overall, this study is the most comprehensive quantitative analysis of the developing brain proteome to date providing an important resource for neurobiologists.

Mass spectrometric strategies to identify protein subpopulations involved in specific biological functions rely on covalently tagging biotin to proteins using various chemical modification methods. The biotin tag is primarily used for enrichment of the targeted subpopulation for subsequent mass spectrometry (MS) analysis. A limitation of these strategies is that MS analysis does not easily discriminate unlabeled contaminants from the labeled protein subpopulation under study. To solve this problem, we developed a flexible method that only relies on direct MS detection of biotin-tagged proteins called “Direct Detection of Biotin-containing Tags” (DiDBiT). Compared with conventional targeted proteomic strategies, DiDBiT improves direct detection of biotinylated proteins ∼200 fold. We show that DiDBiT is applicable to several protein labeling protocols in cell culture and in vivo using cell permeable NHS-biotin and incorporation of the noncanonical amino acid, azidohomoalanine (AHA), into newly synthesized proteins, followed by click chemistry tagging with biotin. We demonstrate that DiDBiT improves the direct detection of biotin-tagged newly synthesized peptides more than 20-fold compared to conventional methods. With the increased sensitivity afforded by DiDBiT, we demonstrate the MS detection of newly synthesized proteins labeled in vivo in the rodent nervous system with unprecedented temporal resolution as short as 3 h.

Large-scale proteomic analysis of the mammalian brain has been successfully performed with mass spectrometry techniques, such as Multidimensional Protein Identification Technology (MudPIT), to identify hundreds to thousands of proteins. Strategies to efficiently quantify protein expression levels in the brain in a large-scale fashion, however, are lacking. Here, we demonstrate a novel quantification strategy for brain proteomics called SILAM (Stable Isotope Labeling in Mammals). We utilized a 15 N metabolically labeled rat brain as an internal standard to perform quantitative MudPIT analysis on the synaptosomal fraction of the cerebellum during post-natal development. We quantified the protein expression level of 1138 proteins in four developmental time points, and 196 protein alterations were determined to be statistically significant. Over 50% of the developmental changes observed have been previously reported using other protein quantification techniques, and we also identified proteins as potential novel regulators of neurodevelopment. We report the first large-scale proteomic analysis of synaptic development in the cerebellum, and we demonstrate a useful quantitative strategy for studying animal models of neurological disease.