Enabling global analysis of protein citrullination and homocitrullination via biotin thiol tag-assisted mass spectrometry

Citrullination and homocitrullination are key post-translational modifications (PTMs) that affect protein structures and functions. Although they have been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize these PTMs. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of these two PTMs simultaneously via mass spectrometry. We perform global mapping of the citrullination and homocitrullination proteomes of mouse tissues. In total, we identify 691 citrullination sites and 81 homocitrullination sites from 432 and 63 proteins, respectively, representing the largest datasets to date. We discover novel distribution and functions of these two PTMs. We also perform multiplexing quantitative analysis via isotopic labeling techniques. This study depicts a landscape of protein citrullination and homocitrullination and lays the foundation for further deciphering their physiological and pathological roles.

Bacillimidazoles A−F, Imidazolium-Containing Compounds Isolated from a Marine Bacillus

Chemical investigations of a marine sponge-associated Bacillus revealed six new imidazolium-containing compounds, bacillimidazoles A–F (1–6). Previous reports of related imidazolium-containing natural products are rare. Initially unveiled by timsTOF (trapped ion mobility spectrometry) MS data, extensive HRMS and 1D and 2D NMR analyses enabled the structural elucidation of 1–6. In addition, a plausible biosynthetic pathway to bacillimidazoles is proposed based on isotopic labeling experiments and invokes the highly reactive glycolytic adduct 2,3-butanedione. Combined, the results of structure elucidation efforts, isotopic labeling studies and bioinformatics suggest that 1–6 result from a fascinating intersection of primary and secondary metabolic pathways in Bacillus sp. WMMC1349. Antimicrobial assays revealed that, of 1–6, only compound six displayed discernible antibacterial activity, despite the close structural similarities shared by all six natural products.

Solid-State 17O NMR Study of α-D-Glucose: Exploring New Frontiers in Isotopic Labeling, Sensitivity Enhancement, and NMR Crystallography

We report synthesis and solid-state 17O NMR characterization of α-D-glucose for which all six oxygen atoms are site-specifically 17O-labeled. Solid-state 17O NMR spectra were recorded for α-D-glucose/NaCl/H2O (2/1/1) cocrystals under...

A workflow to investigate the impacts of weathered multi-walled carbon nanotubes to the mud snail Lymnaea stagnalis

Although the development and application of nanomaterials is a growing industry, little data is available on the ecotoxicological effects on aquatic organisms. Therefore, we set up a workflow to address the potential uptake of weathered multi-walled carbon nanotubes (wMWCNTs) by a model organism, the pulmonary mud snail Lymnaea stagnalis (L. stagnalis), which plays an important role in the food web. It represents a suitable organism for this approach because as a grazer it potentially ingests large amounts of sedimented wMWCNTs. As food source for L. stagnalis, benthic biofilm was investigated by the use of a transmission electron microscope (TEM) and a scanning electron microscope (SEM) after exposure with wMWCNTs. In addition, isotopic labeling was applied with 14C-wMWCNTs (0.1 mg/L) to quantify fate, behavior, and enrichment of 14C-wMWCNTs in benthic biofilm and in L. stagnalis. Enrichment in benthic biofilm amounted to 529.0 µg wMWCNTs/g dry weight and in L. stagnalis to 79.6 µg wMWCNTs/g dry weight. A bioconcentration factor (BCF) for L. stagnalis was calculated (3500 L/kg). We demonstrate the accumulation of wMWCNTs (10 mg/L) in the digestive tract of L. stagnalis in an effect study. Moreover, the physiological markers glycogen and triglycerides as indicators for the physiological state, as well as the RNA/DNA ratio as growth indicator, were examined. No significant differences between exposed and control animals were analyzed for glycogen and triglycerides after 24 days of exposure, but a decreasing trend is recognizable for triglycerides. In contrast, the significant reduction in the RNA/DNA ratio of L. stagnalis indicated an inhibition of growth with a following recovery after depuration. The described workflow enables a comprehensive determination of the fate and the behavior of wMWCNTs specifically and in general all kinds of CNTs in the aquatic environment and therefore contributes to a holistic risk assessment of wMWCNTs.