Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures

AbstractThe structure of N-linked glycans is determined by a complex, anabolic, intracellular pathway but the exact role of individual glycans is not always clear. Characterization of carbohydrates attached to glycoproteins is essential to aid understanding of this complex area of biology. Specific mass spectral detection of glycopeptides from protein digests may be achieved by on-line HPLC-MS, with selected ion monitoring (SIM) for diagnostic product ions generated by cone voltage fragmentation, or by precursor ion scanning for terminal saccharide product ions, which can yield the same information more rapidly. When glycosylation is heterogeneous, however, these approaches can result in spectra that are complex and poorly resolved. We have developed methodology, based around precursor ion scanning for ions of high m/z, that allows site specific detection and structural characterization of glycans at high sensitivity and resolution. These methods have been developed using the standard glycoprotein, fetuin, and subsequently applied to the analysis of the N-linked glycans attached to the scrapie-associated prion protein, PrPSc. These glycans are highly heterogeneous and over 30 structures have been identified and characterized site specifically. Product ion spectra have been obtained on many glycopeptides confirming structure assignments. The glycans are highly fucosylated and carry Lewis X or sialyl Lewis X epitopes and the structures are in-line with previous results. [Abbreviations: Hex–Hexose, C6H12O6 carbohydrates, including mannnose and galactose; HexNAc—N-acetylhexosamine, C8H15NO6 carbohydrates, including N-acetylglucosamine and N-acetylgalactosamine; GlcNAc—N-acetylglucosamine; GalNAc—N-acetylgalactosamine; Fuc–Fucose; NeuAC—N-acetylneuraminic acid or sialic acid; TSE—Transmissible Spongiform Encephalopathy.

Out of the Mouth of Babes: Lessons from Research on Human Infants

Marine mammal behavior and cognition researchers often face a number of challenges, including the research subjects’ lack of interest and verbal abilities, as well as choosing a paradigm with appropriate stimuli for the subjects’ perceptual and cognitive abilities. Researchers who work with human infants often encounter similar challenges when studying infant cognition and have developed strategies to overcome these challenges, including using stimuli that capture the infants’ attention, determining what tasks are age-appropriate, and using conditioned responses to test discrimination abilities. This paper encourages marine mammal researchers to learn from the research paradigms and techniques used in human infant research and alter them appropriately for the intended study subjects. The conditioned head-turn response, Violation-of-Expectation paradigm, and the help/hinder paradigm have all been used in infant cognition research and show great promise for furthering the current understanding of marine mammal behavior and cognition. In addition, studying a subject’s spontaneous behavior can provide valuable insight in areas such as problem solving skills, creativity, and individual differences. Care must be taken to adapt the paradigms and use stimuli to fit each species’ perceptual abilities. For example, avoiding a task that requires color discrimination for species that do not possess color vision or using stimuli that fall within a particular species’ hearing range are necessary steps in designing an ecologically valid and informative study. Adapting paradigms previously used with human infants can help expand the current understanding of marine mammal communication, cognitive abilities, and social behavior

DNA cruciform arms nucleate through a correlated but non-synchronous cooperative mechanism

Inverted repeat (IR) sequences in DNA can form non-canonical cruciform structures to relieve torsional stress. We use Monte Carlo simulations of a recently developed coarse-grained model of DNA to demonstrate that the nucleation of a cruciform can proceed through a cooperative mechanism. Firstly, a twist-induced denaturation bubble must diffuse so that its midpoint is near the centre of symmetry of the IR sequence. Secondly, bubble fluctuations must be large enough to allow one of the arms to form a small number of hairpin bonds. Once the first arm is partially formed, the second arm can rapidly grow to a similar size. Because bubbles can twist back on themselves, they need considerably fewer bases to resolve torsional stress than the final cruciform state does. The initially stabilised cruciform therefore continues to grow, which typically proceeds synchronously, reminiscent of the S-type mechanism of cruciform formation. By using umbrella sampling techniques we calculate, for different temperatures and superhelical densities, the free energy as a function of the number of bonds in each cruciform along the correlated but non-synchronous nucleation pathways we observed in direct simulations.Comment: 12 pages main paper + 11 pages supplementary dat

Large Scale Structures a Gradient Lines: the case of the Trkal Flow

A specific asymptotic expansion at large Reynolds numbers (R)for the long wavelength perturbation of a non stationary anisotropic helical solution of the force less Navier-Stokes equations (Trkal solutions) is effectively constructed of the Beltrami type terms through multi scaling analysis. The asymptotic procedure is proved to be valid for one specific value of the scaling parameter,namely for the square root of the Reynolds number (R).As a result large scale structures arise as gradient lines of the energy determined by the initial conditions for two anisotropic Beltrami flows of the same helicity.The same intitial conditions determine the boundaries of the vortex-velocity tubes, containing both streamlines and vortex linesComment: 27 pages, 2 figure

The effect of organelle discovery upon sub-cellular protein localisation.

Prediction of protein sub-cellular localisation by employing quantitative mass spectrometry experiments is an expanding field. Several methods have led to the assignment of proteins to specific subcellular localisations by partial separation of organelles across a fractionation scheme coupled with computational analysis. Methods developed to analyse organelle data have largely employed supervised machine learning algorithms to map unannotated abundance profiles to known protein–organelle associations. Such approaches are likely to make association errors if organelle-related groupings present in experimental output are not included in data used to create a protein–organelle classifier. Currently, there is no automated way to detect organelle-specific clusters within such datasets. In order to address the above issues we adapted a phenotype discovery algorithm, originally created to filter image-based output for RNAi screens, to identify putative subcellular groupings in organelle proteomics experiments. We were able to mine datasets to a deeper level and extract interesting phenotype clusters for more comprehensive evaluation in an unbiased fashion upon application of this approach. Organelle-related protein clusters were identified beyond those sufficiently annotated for use as training data. Furthermore, we propose avenues for the incorporation of observations made into general practice for the classification of protein–organelle membership from quantitative MS experiments. Biological significance Protein sub-cellular localisation plays an important role in molecular interactions, signalling and transport mechanisms. The prediction of protein localisation by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavour in improving protein annotation. Several such approaches use gradient-based separation of cellular organelle content to measure relative protein abundance across distinct gradient fractions. The distribution profiles are commonly mapped in silico to known protein–organelle associations via supervised machine learning algorithms, to create classifiers that associate unannotated proteins to specific organelles. These strategies are prone to error, however, if organelle-related groupings present in experimental output are not represented, for example owing to the lack of existing annotation, when creating the protein–organelle mapping. Here, the application of a phenotype discovery approach to LOPIT gradient-based MS data identifies candidate organelle phenotypes for further evaluation in an unbiased fashion. Software implementation and usage guidelines are provided for application to wider protein–organelle association experiments. In the wider context, semi-supervised organelle discovery is discussed as a paradigm with which to generate new protein annotations from MS-based organelle proteomics experiments. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012]

Non-Equilibrium Reaction Rates in the Macroscopic Chemistry Method for DSMC Calculations

The Direct Simulation Monte Carlo (DSMC) method is used to simulate the flow of rarefied gases. In the Macroscopic Chemistry Method (MCM) for DSMC, chemical reaction rates calculated from local macroscopic flow properties are enforced in each cell. Unlike the standard total collision energy (TCE) chemistry model for DSMC, the new method is not restricted to an Arrhenius form of the reaction rate coefficient, nor is it restricted to a collision cross-section which yields a simple power-law viscosity. For reaction rates of interest in aerospace applications, chemically reacting collisions are generally infrequent events and, as such, local equilibrium conditions are established before a significant number of chemical reactions occur. Hence, the reaction rates which have been used in MCM have been calculated from the reaction rate data which are expected to be correct only for conditions of thermal equilibrium. Here we consider artificially high reaction rates so that the fraction of reacting collisions is not small and propose a simple method of estimating the rates of chemical reactions which can be used in the Macroscopic Chemistry Method in both equilibrium and non-equilibrium conditions. Two tests are presented: (1) The dissociation rates under conditions of thermal non-equilibrium are determined from a zero-dimensional Monte-Carlo sampling procedure which simulates ‘intra-modal’ non-equilibrium; that is, equilibrium distributions in each of the translational, rotational and vibrational modes but with different temperatures for each mode; (2) The 2-D hypersonic flow of molecular oxygen over a vertical plate at Mach 30 is calculated. In both cases the new method produces results in close agreement with those given by the standard TCE model in the same highly nonequilibrium conditions. We conclude that the general method of estimating the non-equilibrium reaction rate is a simple means by which information contained within non-equilibrium distribution functions predicted by the DSMC method can be included in the Macroscopic Chemistry Method

Environmentally co-occurring mercury resistance plasmids are genetically and phenotypically diverse and confer variable context-dependent fitness effects

Plasmids are important mobile elements that can facilitate genetic exchange and local adaptation within microbial communities. We compared the sequences of four co-occurring pQBR-family environmental mercury resistance plasmids and measured their effects on competitive fitness of a Pseudomonas fluorescens SBW25 host, which was isolated at the same field site. Fitness effects of carriage differed between plasmids and were strongly context dependent, varying with medium, plasmid status of competitor and levels of environmental mercury. The plasmids also varied widely in their rates of conjugation and segregational loss. We found that few of the plasmid-borne accessory genes could be ascribed functions, although we identified a putative chemotaxis operon, a type IV pilus-encoding cluster, and a region encoding putative arylsulfatase enzymes, which were conserved across geographically distant isolates. One plasmid, pQBR55, conferred the ability to catabolise sucrose. Transposons, including the mercury resistance Tn5042, appeared to have been acquired by the different pQBR plasmids by recombination, indicating an important role for horizontal gene transfer in the recent evolution of pQBR plasmids. Our findings demonstrate extensive genetic and phenotypic diversity amongst co-occurring members of a plasmid community and suggest a role for environmental heterogeneity in the maintenance of plasmid diversity

Dynamics of fluctuations in an optical analog of the Laval nozzle

Using the analogy between the description of coherent light propagation in a medium with Kerr nonlinearity by means of nonlinear Schr\"odinger equation and that of a dissipationless liquid we propose an optical analogue of the Laval nozzle. The optical Laval nozzle will allow one to form a transonic flow in which one can observe and study a very unusual dynamics of classical and quantum fluctuations including analogue of the Hawking radiation of real black holes. Theoretical analysis of this dynamics is supported by numerical calculations and estimates for a possible experimental setup are presented.Comment: 7 pages, 4 figure

Cosmological Consequences of Slow-Moving Bubbles in First-Order Phase Transitions

In cosmological first-order phase transitions, the progress of true-vacuum bubbles is expected to be significantly retarded by the interaction between the bubble wall and the hot plasma. We examine the evolution and collision of slow-moving true-vacuum bubbles. Our lattice simulations indicate that phase oscillations, predicted and observed in systems with a local symmetry and with a global symmetry where the bubbles move at speeds less than the speed of light, do not occur inside collisions of slow-moving local-symmetry bubbles. We observe almost instantaneous phase equilibration which would lead to a decrease in the expected initial defect density, or possibly prevent defects from forming at all. We illustrate our findings with an example of defect formation suppressed in slow-moving bubbles. Slow-moving bubble walls also prevent the formation of `extra defects', and in the presence of plasma conductivity may lead to an increase in the magnitude of any primordial magnetic field formed.Comment: 10 pages, 7 figures, replaced with typos corrected and reference added. To appear in Phys. Rev.

Antibody mediated neutralization of myelin associated EphrinB3 accelerates CNS remyelination

This is the final version of the article. It was first available from Springer via http://dx.doi.org/10.1007/s00401-015-1521-1Remyelination in multiple sclerosis (MS) lesions often remains incomplete despite the presence of oligodendrocyte progenitor cells (OPCs). Amongst other factors, successful remyelination depends on the phagocytic clearance of myelin debris. However, the proteins in myelin debris that act as potent and selective inhibitors on OPC differentiation and inhibit CNS remyelination remain unknown. Here, we identify the transmembrane signalling protein EphrinB3 as important mediator of this inhibition, using a protein analytical approach in combination with a primary rodent OPC assay. In the presence of EphrinB3, OPCs fail to differentiate. In a rat model of remyelination, infusion of EphrinB3 inhibits remyelination. In contrast, masking EphrinB3 epitopes using antibodies promotes remyelination. Finally, we identify EphrinB3 in MS lesions and demonstrate that MS lesion extracts inhibit OPC differentiation while antibody-mediated masking of EphrinB3 epitopes promotes it. Our findings suggest that EphrinB3 could be a target for therapies aiming at promoting remyelination in demyelinating disease.This work was supported by the UK MS Society Grant ref: 941/11. MRNK held a NIHR Clinical Lectureship. KAN was supported by an ERC advanced award