On the Reproducibility of Label-Free Quantitative Cross-Linking/Mass Spectrometry

Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS3)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community, we developed a workflow that integrates the established software tools MaxQuant for spectra preprocessing, Xi for cross-linked peptide identification, and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 124 were subsequently quantified across 10 analyses with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides

Estimation of Financial Agent-Based Models with Simulated Maximum Likelihood

This paper proposes a general computational framework for empirical estimation of financial agent based models, for which criterion functions do not have known analytical form. For this purpose, we adapt a nonparametric simulated maximum likelihood estimation based on kernel methods. Employing one of the most widely analysed heterogeneous agent models in the literature developed by Brock and Hommes (1998), we extensively test properties of the proposed estimator and its ability to recover parameters consistently and efficiently using simulations. Key empirical findings point us to the statistical insignificance of the switching coefficient but markedly significant belief parameters defining heterogeneous trading regimes with superiority of trend-following over contrarian strategies. In addition, we document slight proportional dominance of fundamentalists over trend following chartists in main world markets

Neutrally stratified flow modelling over complex terrain at meso-scale: open-cut coal mine study

The objective of this case study was to determine the influence of the coal mine cavity and its vicinity on the main flow field with respect to prevailing wind direction. In order to model the neutrally stratified flow over extremely huge open-cut coal mine Libouš situated at north-west of Czech Republic the laminar flow analogy was applied. The model was scaled down to 1:9000 according to dimensions of wind-tunnel and selected coal mine surroundings. The measurements of longitudinal and lateral velocity components in selected vertical, resp. horizontal planes were performed by LDA. The measured data will be used for validation of CFD simulation and for selections of area of interest at bigger scales where turbulent flow modelling will be performed. The results revealed that not only of cavity shape and deepness but also the surroundings orography has influence on flow pattern, hence on ventilation, within the area of interest

Atmospheric dispersion modelling over complex terrain at small scale

Previous study concerned of qualitative modelling neutrally stratified flow over open-cut coal mine and important surrounding topography at meso-scale (1:9000) revealed an important area for quantitative modelling of atmospheric dispersion at small-scale (1:3300). The selected area includes a necessary part of the coal mine topography with respect to its future expansion and surrounding populated areas. At this small-scale simultaneous measurement of velocity components and concentrations in specified points of vertical and horizontal planes were performed by two-dimensional Laser Doppler Anemometry (LDA) and Fast-Response Flame Ionization Detector (FFID), respectively. The impact of the complex terrain on passive pollutant dispersion with respect to the prevailing wind direction was observed and the prediction of the air quality at populated areas is discussed. The measured data will be used for comparison with another model taking into account the future coal mine transformation. Thus, the impact of coal mine transformation on pollutant dispersion can be observed

Epitope and affinity determination of recombinant Mycobacterium tuberculosis Ag85B antigen towards anti-Ag85 antibodies using proteolytic affinity-mass spectrometry and biosensor analysis

Tuberculosis (TB) is the first cause of death from infectious diseases worldwide. Only a single anti-TB vaccine is currently available for clinical use, but its efficacy is not achieved with certainty. The aim of this work is to provide a basis for the rational design of a neo-glycoconjugate vaccine against TB. Structural characterization of recombinant antigenic proteins from Mycobacterium tuberculosis (MTB) Ag85B (rAg85B, variants, and semi-synthetic glycoconjugates) was initially carried out. Identification of antibody epitope analyses by proteolytic affinity-mass spectrometry and surface plasmon resonance (SPR) biosensor analyses were performed in order to qualitatively identify and quantitatively characterize interaction structures of the antigens with antibodies from different sources. A commercial monoclonal antibody and polyclonal antibodies from different sources (patients with active TB, vaccinated individuals, and a healthy control) were employed to analyze antigen-antibody interactions. These combined approaches provided the identification of different assembled epitope regions on the recombinant MTB antigens, their affinity binding constants in the interactions with specific antibodies, and revealed the importance of protection from excessive glycosylation. The identified epitope peptides should constitute a suitable basis for the design of new specific target vaccines

1.2 A resolution crystal structure of Escherichia coli WrbA holoprotein

Acta Crystallographica Section D: Biological Crystallography. Volume 69, Issue 9, September 2013, Pages 1748-1757.The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2Å resolution, the highest yet reported. Faster-growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only ∼1.6Å resolution and are not analysed further here. The 1.2Å resolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen-bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near-atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out. © 2013 International Union of Crystallography Printed in Singapore-all rights reserved