Policy Review: Addressing the Complex Challenges of Regulating Biotherapeutics

The advancing industry of biotherapeutics is providing the public with new promising and innovative drugs which may pose risks if their production, distribution, and marketing are not directly governed by legislation. Apart from international agreements, such as the Cartagena Protocol, there are no specific and direct laws or regulations governing manipulated cell-based therapeutics in Canada. The introduction of these laws and regulations in Canada will allow for the safe research and use of biotherapeutics in a proactive manner

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results

Advancing Techniques of Structural Mass Spectrometry for Integrative Structural Modelling

Proteins are the fundamental functional units underlying all cellular activities. Protein function emerges from structure. To understand cellular activity and the diseases that arise from protein dysfunction we require knowledge of protein structure and structural dynamics. The toolbox offered by mass spectrometry (MS) allows a wide range of perspectives on protein structure, enabled by the application of chemical reagents that can encode structural properties. Improvements in the performance of labelling chemistries in turn can enhance the data returned and the structural models that are ultimately produced. Photogenerated carbenes are one such high-performance chemistry that offer unbiased sampling of protein structure at timescales relevant to protein dynamics. On the other hand, no single method can offer the breadth of data necessary to produce a comprehensive model of protein structure and dynamics—protein systems span broad spatial and temporal scales that exceed the scope of any single technique. To resolve large and complex protein systems, the integration of multiple data sets from orthogonal techniques is necessary. Here, I evaluate and advance structural MS methods with the goal of improving the accuracy and precision of structural models produced by MS-driven integrative structural modelling. Of particular interest is the application of carbene-based crosslinking and covalent labelling reagents which are shown to produce data with greater sequence coverage and improved accuracy in representing the equilibrated conformational state. Novel analytical software routines are developed to overcome the complications that arise from the labelling of proteins with a non-specific chemistry such as ambiguity in localizing modifications. Structural models are produced with integrative modelling workflows, including the development of a novel modelling restraint based on crosslinking and hydrogen/deuterium exchange data. MS-driven integrative modelling is demonstrated on multiple systems, including large complexes and systems with substantial disorder

Simultaneous Proteoform Analysis of Histones H3 and H4 with a Simplified Middle-Down Proteomics Method

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer “middle-down” protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography–tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis

Mechanism of O(³P) Formation from a Hydroxyl Radical Pair in Aqueous Solution

The reaction mechanism for the rapid formation of a triplet oxygen atom, O­(³P), from a pair of triplet-state hydroxyl radicals in liquid water is explored utilizing extensive Car–Parrinello MD simulations and advanced visualization techniques. The local solvation structures, the evolution of atomic charges, atomic separations, spin densities, electron localization functions, and frontier molecular orbitals, as well as free energy profiles, evidence that the reaction proceeds through a hybrid (hydrogen atom transfer and electron–proton transfer) and hemibond-assisted reaction mechanism. A benchmarking study utilizing high-level <i>ab initio</i> calculations to examine the interactions of a hydroxyl radical pair in the gas phase and the influence of a hemibonded water is also provided. The results presented here should serve as a foundation for further experimental and theoretical studies aimed at better understanding the role and potential applications of the triplet oxygen atom as a potent reactive oxygen species

Structure of the mycobacterial ESX-5 type VII secretion system pore complex

The ESX-5 type VII secretion system is a membrane-spanning protein complex key to the virulence of mycobacterial pathogens. However, the overall architecture of the fully assembled translocation machinery and the composition of the central secretion pore have remained unknown. Here, we present the high-resolution structure of the 2.1-megadalton ESX-5 core complex. Our structure captured a dynamic, secretion-competent conformation of the pore within a well-defined transmembrane section, sandwiched between two flexible protein layers at the cytosolic entrance and the periplasmic exit. We propose that this flexibility endows the ESX-5 machinery with large conformational plasticity required to accommodate targeted protein secretion. Compared to known secretion systems, a highly dynamic state of the pore may represent a fundamental principle of bacterial secretion machineries