Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry

Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community

Effect of microneedles on transdermal permeation enhancement of amlodipine

The present study aimed to investigate the effect of microneedle (MN) geometry parameters like length, density, shape and type on transdermal permeation enhancement of amlodipine (AMLO). Two types of MN devices viz. AdminPatch® arrays (ADM) (0.6, 1.2 and 1.5 mm lengths) and laboratory-fabricated polymeric MNs (PM) of 0.6 mm length were employed. In the case of PMs, arrays were applied thrice at different places within a 1.77-cm2 skin area (PM-3) to maintain the MN density closer to 0.6 mm ADM. Scaling analyses were done using dimensionless parameters like concentration of AMLO (Ct/Cs), thickness (h/L) and surface area of the skin (Sa/L2). Microinjection moulding technique was employed to fabricate PM. Histological studies revealed that the PM, owing to their geometry/design, formed wider and deeper microconduits when compared to ADM of similar length. Approximately 6.84- and 6.11-fold increase in the cumulative amount (48 h) of AMLO permeated was observed with 1.5 mm ADM and PM-3 treatments respectively, when compared to passive permeation amounts. Good correlations (R2 > 0.89) were observed between different dimensionless parameters with scaling analyses. The enhancement in AMLO permeation was found to be in the order of 1.5 mm ADM ≥ PM-3 > 1.2 mm ADM > 0.6 mm ADM ≥PM-1 > passive. The study suggests that MN application enhances the AMLO transdermal permeation and the geometrical parameters of MNs play an important role in the degree of such enhancement

Urinary antihypertensive drug metabolite screening using molecular networking coupled to high-resolution mass spectrometry fragmentation

Introduction Mass spectrometry is the current technique of choice in studying drug metabolism. High-resolution mass spectrometry in combination with MS/MS gas-phase experiments has the potential to contribute to rapid advances in this field. However, the data emerging from such fragmentation spectral files pose challenges to downstream analysis, given their complexity and size. Objectives This study aims to detect and visualize antihypertensive drug metabolites in untargeted metabolomics experiments based on the spectral similarity of their fragmentation spectra. Furthermore, spectral clusters of endogenous metabolites were also examined. Methods Here we apply a molecular networking approach to seek drugs and their metabolites, in fragmentation spectra from urine derived from a cohort of 26 patients on antihypertensive therapy. The mass spectrometry data was collected on a Thermo Q-Exactive coupled to pHILIC chromatography using data dependent analysis (DDA) MS/MS gas-phase experiments. Results In total, 165 separate drug metabolites were found and structurally annotated (17 by spectral matching and 122 by classification based on a clustered fragmentation pattern). The clusters could be traced to 13 drugs including the known antihypertensives verapamil, losartan and amlodipine. The molecular networking approach also generated clusters of endogenous metabolites, including carnitine derivatives, and conjugates containing glutamine, glutamate and trigonelline. Conclusions The approach offers unprecedented capability in the untargeted identification of drugs and their metabolites at the population level and has great potential to contribute to understanding stratified responses to drugs where differences in drug metabolism may determine treatment outcome

Field performance of the Chemcatcher passive sampler for monitoring hydrophobic organic pollutants in surface water

Six field trials were carried out to assess the performance of the Chemcatcher passive sampler alongside spot sampling for monitoring priority hydrophobic organic pollutants (polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides) in a wide range of conditions in surface water. The trials were performed in three European rivers: Elbe (Czech Republic), Alna (Norway) and Meuse (Netherlands), in two seasons (April-June 2004, and September-October 2004). Samplers spiked with performance reference compounds (PRCs) were deployed for either 14 or 28 days. Ten spot samples of water were collected over the course of the trial and filtered through a 0.7 mu m glass fibre filter. Concentrations of pollutants measured using the Chemcatcher were compared with the average concentrations found in spot samples. This study describes the operational performance of Chemcatcher for measuring hydrophobic (log K-OW 3.7-6.8) chemicals in surface water. Site specific Chemcatcher sampling rates up to 0.5 L d(-1) were found using the PRC approach that reduced the uncertainty in estimates of sampling kinetics where temperature, local flow conditions and biofouling potential varied between sites and seasons, and with time during sampler exposure. The limits of quantification of sampled analytes ranged from one to tens ng L-1. Highest sensitivity was achieved for compounds with a favourable combination of low instrument quantification limits and high sampling rates including dieldrin, hexachlorobenzene, lindane, pentachlorobenzene, and PAHs with less than five aromatic rings. The direct comparison of time weighted average (TWA) concentrations (mostly close to method limits of detection) obtained using passive and spot sampling was possible for lindane, hexachlorobenzene, and PAHs <4 rings. Implications of using the Chemcatcher in regulatory monitoring programmes such as the European Union Water Framework Directive are discussed

The Strategies of Translating Economics and Business Terms from English into Lithuanian

The problems of translating the economics and business terms from English into Lithuanian, associated with the choice of particular translation strategies are considered based on the analysis of the translation of the English Economics Dictionary into the Lithuanian language. The classification of the translation strategies into domesticating and foreignizing methods and the description of other translating techniques are presented, and their advantages and disadvantages are described, taking into account the status of particular languages. The main strategies used in translating the considered Dictionary are determined, and their strengths and weaknesses are analysed from various perspectives. The analysis is based on the established structural-semantic models, underlying the formation of the parallel English and Lithuanian terms presented in the considered dictionary, and determining their similar and different features. The method of the translation changes characteristic of the foreignizing strategy of translation is also analysed. The prospects of further research required for checking the practical use of the translated economics and business terms in Lithuanian works in these fields are also outlined. It is also emphasized that the methods used and the results obtained in the present investigation may be used as a basis for further studies

HDX-ESI-MS Reveals Enhanced Conformational Dynamics of the Amyloidogenic Protein β2-Microglobulin upon Release from the MHC-1

The light chain of the major histocompatibility complex class 1 (MHC-1), the protein β2-microglobulin (β2m), has amyloidogenic properties that arise only upon its dissociation from the MHC-1. Here hydrogen/deuterium exchange electrospray ionization mass spectrometry (HDX-ESI-MS) has been used to compare the solution dynamics of β2m in its MHC-1 bound state compared with those of β2m as a free monomer. The capability of tandem mass spectrometry to dissociate the MHC-1 into its individual constituents in the gas phase following deuterium incorporation in solution has permitted the direct observation of the exchange properties of MHC-1 bound β2m for the first time. The HDX-ESI-MS data show clearly that the H→D exchange of MHC-1 bound β2m follows EX2 kinetics and that about 20 protons remain protected from exchange after 17 days. Free from the MHC-1, monomeric β2m exhibits significantly different HDX behavior, which encompasses both EX1 and EX2 kinetics. The EX2 kinetics indicate a tenfold increase in the rate of exchange compared with MHC-1 bound β2m, with just 10 protons remaining protected from EX2 exchange and therefore exchanging only via the EX1 mechanism. The EX1 kinetics observed for unbound β2m are consistent with unfolding of its exchange-protected core with a t1/2 of 68 min (pH 7, 37° C). Thus, upon dissociation from the stabilizing influence of the MHC-1, free β2m becomes highly dynamic and undergoes unfolding transitions that result in an aggregation-competent protein