Local Ca 2+ signaling occurring within nanometers of voltage-gated Ca 2+ (Cav) channels is crucial for CNS function, yet the molecular composition of Cav channel nano-environments is largely unresolved. Here, we used a proteomic strategy combining knockoutcontrolled multiepitope affinity purifications with high-resolution quantitative MS for comprehensive analysis of the molecular nano-environments of the Cav2 channel family in the whole rodent brain. The analysis shows that Cav2 channels, composed of poreforming α1 and auxiliary β subunits, are embedded into protein networks that may be assembled from a pool of ∼200 proteins with distinct abundance, stability of assembly, and preference for the three Cav2 subtypes. The majority of these proteins have not previously been linked to Cav channels; about two-thirds are dedicated to the control of intracellular Ca 2+ concentration, including G proteincoupled receptor-mediated signaling, to activity-dependent cytoskeleton remodeling or Ca 2+ -dependent effector systems that comprise a high portion of the priming and release machinery of synaptic vesicles. The identified protein networks reflect the cellular processes that can be initiated by Cav2 channel activity and define the molecular framework for organization and operation of local Ca 2+ signaling by Cav2 channels in the brain.calcium channel | Ca 2+ signaling | proteome | biochemistry | mass spectrometry

Native AMPA receptors (AMPARs) in the mammalian brain are macromolecular complexes whose functional characteristics vary across the different brain regions and change during postnatal development or in response to neuronal activity. The structural and functional properties of the AMPARs are determined by their proteome, the ensemble of their protein building blocks. Here we use high-resolution quantitative mass spectrometry to analyze the entire pool of AMPARs affinity-isolated from distinct brain regions, selected sets of neurons, and whole brains at distinct stages of postnatal development. These analyses show that the AMPAR proteome is dynamic in both space and time: AMPARs exhibit profound region specificity in their architecture and the constituents building their core and periphery. Likewise, AMPARs exchange many of their building blocks during postnatal development. These results provide a unique resource and detailed contextual data sets for the analysis of native AMPAR complexes and their role in excitatory neurotransmission.

Affinity purification (AP) of protein complexes combined with LC-MS/MS analysis is the current method of choice for identification of protein-protein interactions. Their interpretation with respect to significance, specificity, and selectivity requires quantification methods coping with enrichment factors of more than 1000-fold, variable amounts of total protein, and low abundant, unlabeled samples. We used standardized samples (0.1-1000 fmol) measured on high resolution hybrid linear ion trap instruments (LTQ-FT/Orbitrap) to characterize and improve linearity and dynamic range of label-free approaches. Quantification based on spectral counts was limited by saturation and ion suppression effects with samples exceeding 100 ng of protein, depending on the instrument setup. In contrast, signal intensities of peptides (peak volumes) selected by a novel correlation-based method (TopCorr-PV) were linear over at least 4 orders of magnitude and allowed for accurate relative quantification of standard proteins spiked into a complex protein background. Application of this procedure to APs of the voltage-gated potassium channel Kv1.1 as a model membrane protein complex unambiguously identified the whole set of known interaction partners together with novel candidates. In addition to discriminating these proteins from background, we could determine efficiency, cross-reactivities, and selection biases of the used purification antibodies. The enhanced dynamic range of the developed quantification procedure appears well suited for sensitive identification of specific protein-protein interactions, detection of antibody-related artifacts, and optimization of AP conditions. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.007955, 1-12, 2012.Antibody-based affinity purification (AP) 1 of protein assemblies from biological samples followed by mass spectrometric analysis represents an increasingly popular approach for identification of protein-protein interactions (AP-MS) (1-3). Despite the exquisitely high and specific enrichment theoretically obtainable with antibodies (Abs), this approach faces a number of technical and intrinsic challenges in practice. Target protein complexes typically suffer from poor solubility, instability, and low abundance, particularly when associated with lipid membranes. Moreover, various antibody-related properties such as target selectivity, cross-reactivity, and interference with protein-protein interactions may lead to falsepositive and false-negative results (4). Finally, biological protein-protein interactions may have a more dynamic character, may depend on regulated modifications, or may involve rare protein partners. Together, these effects lead to a significant reduction of AP signal to noise, i.e. low co-enrichment efficiency of interaction partners and significant overlap with background or nonspecific proteins.Classically, AP specificity has been addressed by visualization of purified proteins on one-or two-dimensional gels and comparison of band patterns or spots with those obtained in...