Mass spectrometry (MS)-based proteomics is emerging as a broadly effective means for identification, characterization, and quantification of proteins that are integral components of the processes essential for life. Characterization of proteins at the proteome and sub-proteome (e.g., the phosphoproteome, proteoglycome, or degradome/peptidome) levels provides a foundation for understanding fundamental aspects of biology. Emerging technologies such as ion mobility separations coupled with MS and microchip-based-proteome measurements combined with MS instrumentation and chromatographic separation techniques, such as nanoscale reversed phase liquid chromatography and capillary electrophoresis, show great promise for both broad undirected and targeted highly sensitive measurements. MS-based proteomics is increasingly contribute to our understanding of the dynamics, interactions, and roles that proteins and peptides play, advancing our understanding of biology on a systems wide level for a wide range of applications including investigations of microbial communities, bioremediation, and human health.

G protein-coupled receptors with seven transmembrane ␣-helices (GPCRs) comprise the largest receptor superfamily and are involved in detecting a wide variety of extracellular stimuli. The availability of high-resolution crystal structures of five prototypical GPCRs, bovine and squid rhodopsin, engineered A 2A-adenosine, ␤1-and ␤2-adrenergic receptors, permits comparative analysis of features common to these and likely all GPCRs. We provide an analysis of the distribution of water molecules in the transmembrane region of these GPCR structures and find conserved contacts with microdomains demonstrated to be involved in receptor activation. Colocalization of water molecules associating with highly conserved and functionally important residues in several of these GPCR crystal structures supports the notion that these waters are likely to be as important to proper receptor function as the conserved residues. Moreover, in the absence of large conformational changes in rhodopsin after photoactivation, we propose that ordered waters contribute to the functional plasticity needed to transmit activation signals from the retinal-binding pocket to the cytoplasmic face of rhodopsin and that fundamental features of the mechanism of activation, involving these conserved waters, are shared by many if not all family A receptors.adrenergic receptor ͉ allosteric modulator ͉ ordered waters ͉ transmembrane proteins ͉ x-ray G protein-coupled receptors (GPCRs) are membrane proteins that mediate signal transduction by recognizing a wide range of extracellular stimuli ranging from photons of light, biogenic amines and odorants, to small peptides that all induce receptor activation. Activated GPCRs signal through heterotrimeric G proteins to activate effector enzymes, resulting in rapid signal amplification. Given the new-found wealth of structural information available from determination of several GPCR crystal structures, our purpose here is to provide a structural analysis of components likely to participate in the allosteric communication between the ligand-binding site near the extracellular domain and the cytoplasmic site involved in G protein coupling and activation.The first high-resolution crystal structure of a GPCR was that of bovine rhodopsin (1), an advance that facilitated the successful determination of other high-resolution bovine rhodopsin structures (2-5). One paradigm for GPCR activation involves large rigid body movements of up to 15 Å of the heptahelical transmembrane bundle upon receptor transition from an inactive to an active conformation (6). This notion has been challenged because large changes in these helices were not observed in the crystal structure of a late photointermediate of rhodopsin that retained the fullagonist, all-trans-retinylidene, and exhibited spectral features consistent with the metarhodopsin (Meta) II activated state (7). Newer studies using double electron-electron resonance (DEER) spectroscopy revised the predicted structural changes upon activation to be no more than 5 Å between helices (...

BackgroundKnowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal.Methods and Principal FindingsWe applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.ConclusionsOur results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features.

Structural water molecules may act as prosthetic groups indispensable for proper protein function. In the case of allosteric activation of G protein-coupled receptors (GPCRs), water likely imparts structural plasticity required for agonist-induced signal transmission. Inspection of structures of GPCR superfamily members reveals the presence of conserved embedded water molecules likely important to GPCR function. Coupling radiolytic hydroxyl radical labeling with rapid H 2O 18 solvent mixing, we observed no exchange of these structural waters with bulk solvent in either ground state or for the Meta II or opsin states. However, the radiolysis approach permitted labeling of selected side chain residues within the transmembrane helices and revealed activation-induced changes in local structural constraints likely mediated by dynamics of both water and protein. These results suggest both a possible general mechanism for water-dependent communication in family A GPCRs based on structural conservation, and a strategy for probing membrane protein structure.footprinting ͉ mass spectrometry ͉ signal transduction ͉ membrane proteins ͉ radiolysis

The structures of protein antigen–antibody (Ag–Ab) interfaces contain information about how Ab recognize Ag as well as how Ag are folded to present surfaces for Ag recognition. As such, the Ab surface holds information about Ag folding that resides with the Ab–Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, a data set comprised of 53 non-redundant 3D structures of Ag–Ab complexes was analyzed. We assessed the physical and biochemical features of the Ag–Ab interfaces and the degree to which favored interactions exist between amino acid residues on the corresponding interface surfaces. Amino acid compositional analysis of the interfaces confirmed the dominance of TYR in the Ab paratope-containing surface (PCS), with almost two fold greater abundance than any other residue. Additionally TYR had a much higher than expected presence in the PCS compared to the surface of the whole antibody (defined as the occurrence propensity), along with aromatics PHE, TRP, and to a lesser degree HIS and ILE. In the Ag epitope-containing surface (ECS), there were slightly increased occurrence propensities of TRP and TYR relative to the whole Ag surface, implying an increased significance over the compositionally most abundant LYS>ASN>GLU>ASP>ARG. This examination encompasses a large, diverse set of unique Ag–Ab crystal structures that help explain the biological range and specificity of Ag–Ab interactions. This analysis may also provide a measure of the significance of individual amino acid residues in phage display analysis of Ag binding.