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The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a multidomain catalyst of disulfide-bond formation that relays electrons from substrate cysteines through two redox-active sites to molecular oxygen. The chemical steps in electron transfer have been delineated, but the conformational changes accompanying these steps are poorly characterized. Here we use single-molecule Förster resonance energy transfer (smFRET) to probe QSOX conformation in resting and cycling enzyme populations. We report the discovery of unanticipated roles for conformational changes in QSOX beyond mediating electron transfer between redox-active sites. In particular, a state of the enzyme not previously postulated or experimentally detected is shown to gate, via a conformational transition, the entrance into a sub-cycle within an expanded QSOX kinetic scheme. By tightly constraining mechanistic models, smFRET data can reveal the coupling between conformational and chemical transitions in complex enzymatic cycles. A major challenge in following electron transfer through dithiol/disulfide exchange is the dearth of accompanying spectroscopic effects. Here, the authors use single-molecule Förster resonance energy transfer experiments to illuminate disulfide bond rearrangements within the enzyme quiescin sulfhydryl oxidase.

Extracellular vesicles (EVs) are blood‐borne messengers that coordinate signalling between different tissues and organs in the body. The specificity of such crosstalk is determined by preferential EV docking to target sites, as mediated through protein‐protein interactions. As such, the need to structurally characterize the EV surface precedes further understanding of docking selectivity and recipient‐cell uptake mechanisms. Here, we describe an intact extracellular vesicle crosslinking mass spectrometry (iEVXL) method that can be applied for structural characterization of protein complexes in EVs. By using a partially membrane‐permeable disuccinimidyl suberate crosslinker, proteins on the EV outer‐surface and inside EVs can be immobilized together with their interacting partners. This not only provides covalent stabilization of protein complexes before extraction from the membrane‐enclosed environment, but also generates a set of crosslinking distance restraints that can be used for structural modelling and comparative screening of changes in EV protein assemblies. Here we demonstrate iEVXL as a powerful approach to reveal high‐resolution information, about protein determinants that govern EV docking and signalling, and as a crucial aid in modelling docking interactions.

Background/Objectives: Multiple Myeloma (MM) is a hematologic malignancy caused by clonally expanded plasma cells that produce a monoclonal immunoglobulin (M-protein), a personalized biomarker. Recently, we developed an ultra-sensitive mass spectrometry method to quantify minimal residual disease (MS-MRD) by targeting unique M-protein peptides. Therapeutic antibodies (t-Abs), key in MM treatment, often lead to deep and long-lasting responses. However, t-Abs can significantly decrease the total polyclonal immunoglobulin (Ig) levels which require supplemental IgG infusion. Here, we demonstrate the simultaneous monitoring of M-proteins, t-Abs, and polyclonal Ig-titers using an untargeted mass spectrometry assay, offering a comprehensive view of therapy response. Methods: Sera collected between 2013 and 2024 from four patients and cerebrospinal fluid (CSF) from one patient who received various t-Abs were analyzed with MS-MRD. M-protein sequences were obtained with a multi-enzyme de novo protein sequencing approach. Unique peptides for M-proteins and t-Abs were selected based on linearity, sensitivity, and slope coefficient in serial dilutions. Ig constant regions were monitored using isotype-specific peptides. Results: The MS-MRD multiplex analysis provided detailed information on drug concentrations and therapy response kinetics. For example, in two patients with refractory disease over five lines of therapy, the MS-MRD analysis showed that the deepest responses were achieved with bispecific t-Ab (teclistamab) treatment. M-protein and t-Ab were also detectable in the CSF of one patient with MS-MRD. Conclusions: This proof-of-concept study shows that the multiplex monitoring of the M-protein, any t-Ab combination, and all Ig-isotypes within one mass spectrometry run is feasible and provides unique insight into therapy response kinetics.

Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins.

The two major proteins involved in Alzheimer’s disease (AD) are the amyloid precursor protein (APP) and Tau. Here, we demonstrate that these two proteins can bind to each other. Four possible peptides APP1 (390–412), APP2 (713–730), Tau1 (19–34) and Tau2 (331–348), were predicted to be involved in this interaction, with actual binding confirmed for APP1 and Tau1. In vivo studies were performed in an Alzheimer Disease animal model—APP double transgenic (Tg) 5xFAD—as well as in 5xFAD crossed with Tau transgenic 5xFADXTau (FT), which exhibit declined cognitive reduction at four months of age. Nasal administration of APP1 and Tau1 mixture, three times a week for four or five months, reduced amyloid plaque burden as well as the level of soluble Aβ 1–42 in the brain. The treatment prevented the deterioration of cognitive functions when initiated at the age of three months, before cognitive deficiency was evident, and also at the age of six months, when such deficiencies are already observed, leading to a full regain of cognitive function.

Real-time database searching allows for simpler and automated proteomics workflows as it eliminates technical bottlenecks in high-throughput experiments. Most importantly, it enables results-dependent acquisition (RDA), where search results can be used to guide data acquisition during acquisition. This is especially beneficial for glycoproteomics since the wide range of physicochemical properties of glycopeptides lead to a wide range of optimal acquisition parameters. We established here the GlycoPaSER prototype by extending the Parallel Search Engine in Real-time (PaSER) functionality for real-time glycopeptide identification from fragmentation spectra. Glycopeptide fragmentation spectra were decomposed into peptide and glycan moiety spectra using common N-glycan fragments. Each moiety was subsequently identified by a specialized algorithm running in real-time. GlycoPaSER can keep up with the rate of data acquisition for real-time analysis with similar performance to other glycoproteomics software and produces results that are in line with the literature reference data. The GlycoPaSER prototype presented here provides the first proof-of-concept for real-time glycopeptide identification that unlocks the future development of RDA technology to transcend data acquisition.

Cross-linking analyzed by mass spectrometry (XL-MS) is a new tool for structural biology which is especially useful for studying the organization of protein complexes. In my Ph.D. research, I used XL-MS to obtain structural information on the protein complex laminin. Laminin, a ~800 kD heterotrimeric protein, is a major functional component of the extracellular matrix (ECM), contributing to tissue development and maintenance. The laminin complex has a unique architecture that is currently not amenable to determination at high resolution because its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. XL-MS has been well-suited for determining the organization of the laminin complex, especially to the ~750 Å coiled coil that mediates trimer assembly, due to the abundance of solvent-exposed residues. The cross-linking data, evaluated using novel computational methods, revealed two fundamental aspects of the coiled-coil structure: the subunit order and subunit register. This structural information provided insights into interactions of laminin domains and contacts between laminin and other ECM components. The subunit order and register may facilitate future studies of the physical basis of laminin’s interactions. XL-MS is mostly used on purified protein complexes like laminin, but it has the potential to provide structural information on protein complexes in their native environments. Along these lines, we have used XL-MS on intact complex systems: the giant virus Acanthamoeba polyphaga Mimivirus and intact ECM produced by cultured fibroblasts. We used cross-linking data of the Mimivirus to identify solvent-exposed proteins and a few interactions, most notably the formation of homomultimers. To date, cross-linking data of the ECM have not provided new structural information but are consistent with known domain structures. These results indicate that XL-MS experiments on these complex systems have the potential to yield structural insights; these experiments provide the foundation for improved experiments and expanded data collection.

Real-time database searching allows for simpler and automated proteomics workflows as it eliminates technical bottlenecks in high throughput experiments. Most importantly, it enables results dependent acquisition (RDA) where search results can be used to guide data acquisition during acquisition. This is especially beneficial for glycoproteomics since the wide range of physicochemical properties of glycopeptides lead to a wide range of optimal acquisition parameters. We established here the GlycoPaSER prototype by extending the Parallel Search Engine in Real-time (PaSER) functionality for real-time glycopeptide identification from fragmentation spectra. Glycopeptide fragmentation spectra were decomposed into peptide- and glycan-moiety spectra using common N-glycan fragments. Each moiety was subsequently identified by a specialized algorithm running in real-time. GlycoPaSER can keep up with the rate of data acquisition for real-time analysis with similar performance to other glycoproteomics software and produces results that are in line with literature reference data. The GlycoPaSER prototype presented here provides the first proof-of-concept for real-time glycopeptide identification that unlocks future development of RDA technology to transcend data acquisition.Competing Interest StatementThe appointment of Gad Armony was in part funded by Bruker Daltonics and co-authors Sven Brehmer, Tharan Srikumar, Lennard Pfennig, Dennis Trede, and Gary Kruppa are employees of Bruker Daltonics. Bruker Daltonics is the manufacturer of the mass spectrometry hardware and software platforms used in this work.