There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.

Summary All seven lysine residues in ubiquitin contribute to the synthesis of polyubiquitin chains on protein substrates. Whereas K48-linked chains are well established as mediators of proteasomal degradation, and K63-linked chains act in nonproteolytic events, the roles of unconventional polyubiquitin chains linked through K6, K11, K27, K29, or K33 are not well understood. Here we report that the unconventional linkages are abundant in vivo, and all non-K63 linkages may target proteins for degradation. Ubiquitin with K48 as the single lysine cannot support yeast viability, and different linkages have partially redundant functions. By profiling both the entire yeast proteome and ubiquitinated proteins in wild-type and ubiquitin K11R mutant strains using mass spectrometry, we identified K11 linkage-specific substrates, including Ubc6, a ubiquitin conjugating enzyme involved in endoplasmic reticulum-associated degradation (ERAD). Ubc6 primarily synthesizes K11-linked chains, and K11 linkages function in the ERAD pathway. Thus, unconventional polyubiquitin chains are critical for ubiquitin-proteasome system function.

The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD. Molecular & Cellular Proteomics 5:1158 -1170, 2006.In excitatory synapses of the mammalian brain, the postsynaptic density (PSD) 1 is a specialized membrane-associated structure containing a high concentration of glutamate receptors, cell adhesion molecules, and associated scaffold proteins and signaling enzymes (1-3). Glutamate receptors in the PSD are assembled into large protein complexes by binding to PDZ domain-containing scaffold proteins. In a well characterized example, the cytoplasmic C termini of NR2 subunits of the NMDA-type glutamate receptor interact with the PDZ domains of the PSD-95 family of scaffold proteins, which are highly enriched in the PSD (2). PSD-95 in turn binds to cytoplasmic signaling proteins such as SynGAP, the synaptic GTPase-activating protein (GAP) for Ras/Rap small GTPases (4, 5). Assembly of such protein complexes facilitates specific coupling of postsynaptic receptors to trafficking mechanisms and downstream signaling pathways that control synaptic strength, cytoskeletal rearrangements, and nuclear responses (1). Many if not most of the protein constituents of the PSD are dynamically influenced by synaptic activity via mechanisms such as protein phosphorylation, local translation, ubiquitination, degradation (6, 7), and protein translocation into and out of synapses (8). Altered composition and structural remodeling of the PSD are believed to play critical roles in the formation/elimination and plasticity of synapses. Because it is amenable to biochemical purification and because of its compact size (a few hundred nanometers in diameter and 20 -40 nm thick), the PSD is a highly suitable "o...

The postsynaptic density (PSD) of central excitatory synapses plays a key role in postsynaptic signal transduction and contains a high concentration of glutamate receptors and associated scaffold and signaling proteins. We report here a comprehensive analysis of purified PSD fractions by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We identified 374 different proteins that copurified with the PSD structure and discovered thirteen phosphorylated sites from eight proteins. These proteins were classified into numerous functional groups, implying that the signaling pathways in the PSD are complex and diverse. Furthermore, using quantitative mass spectrometry, we measured the molar concentration and relative stoichiometries of a number of glutamate receptor subunits and scaffold proteins in the postsynaptic density. Thus this proteomic study reveals crucial information about molecular abundance as well as molecular diversity in the PSD, and provides a basis for further studies on the molecular mechanisms of synaptic function and plasticity.In excitatory synapses of the brain, the postsynaptic density (PSD) 1 is a microdomain of the postsynaptic membrane specialized for signaling and plasticity (1-3). It contains neurotransmitter receptors (particularly for the transmitter glutamate), receptor-associated scaffold proteins, cytoskeletal elements, and regulatory enzymes, which are assembled together in a disk-like structure, ϳ30 -40-nm thick and a few hundred nanometers wide (4). Glutamate receptors in the PSD are organized into supramolecular signaling complexes by interacting with specific PDZ (PSD-95, Dlg, ZO-1 homology) domain-containing scaffolds (such as PSD-95/SAP90) and their associated proteins.The molecular architecture of the PSD is specialized for signal transduction, but it is also highly modifiable to allow for strengthening and weakening of synaptic transmission (1-3). The function and composition of the PSD is dynamically regulated in response to neural activity, involving mechanisms such as protein phosphorylation (3), local translation (5), ubiquitination and degradation (6, 7), and subcellular redistribution (8). Because of the central role of the PSD in synaptic transmission and plasticity, a comprehensive knowledge of the protein composition of the PSD will be extremely useful for understanding synaptic mechanisms, even if this composition represents only an averaged "snapshot" of a heterogeneous population of changing PSDs.Over years, protein components of the PSD have been gradually discovered by biochemical and yeast two-hybrid approaches. The PSD is typically purified through differential centrifugation, sucrose gradient sedimentation and detergent extraction, because the PSD structure cannot be solubilized with mild nonionic detergents such as Triton X-100 (4). Kennedy and co-workers (9, 10) discovered several PSD proteins (e.g. PSD-95, densin-180) by sequencing of protein bands from one-dimensional gels of PSD preparations. Walsh and Kuruc (11) employed two-dimensi...