Oxidation-Induced Protein Cross-Linking in Mammalian Cells.

A proximity-enabled protein cross-linking strategy with additional spatiotemporal control is highly desirable. Here, we report an oxidation-induced protein cross-linking strategy involving the incorporation of a vinyl thioether group into proteins in both Escherichia coli and mammalian cells via genetic code expansion. We demonstrated that vinyl thioether can be selectively induced by exogenously added oxidant or by reactive oxygen species from the cellular environment, as well as by photocatalysts, and converted into a Michael acceptor, enabling fluorescence labeling and protein cross-linking.

Enabling Global Analysis of Protein Citrullination via Biotin Thiol Tag-Assisted Mass Spectrometry.

Citrullination is a key post-translational modification (PTM) that affects protein structures and functions. Although it has 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 this PTM. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of citrullination via mass spectrometry. We perform global mapping of the citrullination proteome of mouse tissues. In total, we identify 691 citrullination sites from 432 proteins which represents the largest data set to date. We discover novel distribution and functions of this PTM. This study depicts a landscape of protein citrullination and lays the foundation for further deciphering their physiological and pathological roles.

Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome.

Lactylation was initially discovered on human histones. Given its nascence, its occurrence on nonhistone proteins and downstream functional consequences remain elusive. Here we report a cyclic immonium ion of lactyllysine formed during tandem mass spectrometry that enables confident protein lactylation assignment. We validated the sensitivity and specificity of this ion for lactylation through affinity-enriched lactylproteome analysis and large-scale informatic assessment of nonlactylated spectral libraries. With this diagnostic ion-based strategy, we confidently determined new lactylation, unveiling a wide landscape beyond histones from not only the enriched lactylproteome but also existing unenriched human proteome resources. Specifically, by mining the public human Meltome Atlas, we found that lactylation is common on glycolytic enzymes and conserved on ALDOA. We also discovered prevalent lactylation on DHRS7 in the draft of the human tissue proteome. We partially demonstrated the functional importance of lactylation: site-specific engineering of lactylation into ALDOA caused enzyme inhibition, suggesting a lactylation-dependent feedback loop in glycolysis.

Time-Resolved Acetaldehyde-Based Accessibility Profiling Maps Ligand-Target Interactions.

Elucidating ligand-protein interactions is important in understanding the biochemical machinery for given proteins. Previously, formaldehyde (FH)-based labeling has been employed to obtain such structural knowledge, since reactive residues that participate in ligand-target interactions display reduced accessibility to FH-labeling reagents, and thus can be identified by quantitative proteomics. Although being rapid and efficient for probing proteinaceous lysine accessibility, here, we report an acetaldehyde (AcH)-labeling approach that complements with FH for probing ligand-target interactions. AcH labeling examines lysine accessibility at a more moderate reaction speed and hence delivers a cleaner reaction when compared to that of FH. The subsequent application of AcH to label RNase A without and with ligands has assisted to assign lysines involved in ligand-RNase A binding by detecting the time-dependent changes in accessibility profiles. We further employed multiple reaction monitoring (MRM) to quantify these ligand-binding-responsive sites when a variety of potential ligands were queried. We noted that the time-resolved abundance changes of these peptides can sensitively determine the ligand-binding sites and differentiate binding affinities among these ligands, which was confirmed by native mass spectrometry (MS) and molecular docking. Lastly, we demonstrated that the binding sites can be recognized by monitoring the chemical accessibility of these responsive peptides in cell lysates. Together, we believe that the proposed combined use of AcH-based lysine accessibility profiling, native MS, and MRM screening is a powerful toolbox in characterizing ligand-target interactions, mapping topography, and interrogating affinities and holds promise for future applications in a complex cellular environment.