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The field of structural biology is increasingly focusing on studying proteins in situ, i.e., in their greater biological context. Cross-linking mass spectrometry (CLMS) is contributing to this effort, typically through the use of mass spectrometry (MS)-cleavable cross-linkers. Here, we apply the popular noncleavable cross-linker disuccinimidyl suberate (DSS) to human mitochondria and identify 5518 distance restraints between protein residues. Each distance restraint on proteins or their interactions provides structural information within mitochondria. Comparing these restraints to protein data bank (PDB)-deposited structures and comparative models reveals novel protein conformations. Our data suggest, among others, substrates and protein flexibility of mitochondrial heat shock proteins. Through this study, we bring forward two central points for the progression of CLMS towards large-scale in situ structural biology: First, clustered conflicts of cross-link data reveal in situ protein conformation states in contrast to error-rich individual conflicts. Second, noncleavable cross-linkers are compatible with proteome-wide studies.

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-β, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-β. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-β, and is distracted from itself., Our understanding on the humoral immunity induced by SARS-CoV-2 is still lacking. Here the authors analyze B cell responses at the single cell level to find that, in severe COVID-19 patients, plasmablasts shift from IFN to TGFβ instruction to produce IgA antibodies that are not specific to dominant SARS-CoV-2 antigens.

Receptor occupancy, the ratio between amount of drug bound and amount of total receptor on single cells, is a biomarker for treatment response to therapeutic monoclonal antibodies. Receptor occupancy is traditionally measured by flow cytometry. However, spectral overlap in flow cytometry limits the number of markers that can be measured simultaneously. This restricts receptor occupancy assays to the analysis of major cell types, although rare cell populations are of potential therapeutic relevance. We therefore developed a receptor occupancy assay suitable for mass cytometry. Measuring more markers than currently available in flow cytometry allows simultaneous receptor occupancy assessment and high‐parameter immune phenotyping in whole blood, which should yield new insights into disease activity and therapeutic effects. However, varying sensitivity across the mass cytometer detection range may lead to misinterpretation of the receptor occupancy when drug and receptor are detected in different channels. In this report, we describe a method for optimization of mass cytometry receptor occupancy measurements by using antibody‐binding quantum simply cellular (QSC) beads for standardization across channels with different sensitivities. We evaluated the method in a mass cytometry‐based receptor occupancy assay for natalizumab, a therapeutic antibody used in multiple sclerosis treatment that binds to α4‐integrin, which is expressed on leukocyte cell surfaces. Peripheral blood leukocytes from a treated patient were stained with a panel containing metal‐conjugated antibodies for detection of natalizumab and α4‐integrin. QSC beads with known antibody binding capacity were stained with the same metal‐conjugated antibodies and were used to standardize the signal intensity in the leukocyte sample before calculating receptor occupancy. We found that QSC bead standardization across channels corrected for sensitivity differences for detection of drug and receptor and generated more accurate results than observed without standardization. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Polystyrene beads are broadly applied in flow cytometry. Implementing bead-based assays in mass cytometry is desired but hampered by the lack of an elemental label required for their detection. In this study, we introduce stable osmium tetroxide labeling as a universal approach for generating functionalized beads readily detectable by mass cytometry. We demonstrate the utility of osmium-labeled beads for signal spillover compensation in mass cytometry, and, strikingly, their application in quantitative Ab-binding capacity assays combined with high-dimensional profiling of human PBMC enabled the systematic assessment of receptor expression profiles across large numbers of cellular phenotypes. This analysis confirmed increased monocytic Siglec-1 expression in active systemic lupus erythematosus patients and, additionally, revealed interrelated reductions of CD4 expression by regulatory and memory CD4 T cells and HLA-DR expression by myeloid dendritic cells, pointing toward defective cross-talk at the immunological synapse that may limit immune responses in systemic lupus erythematosus. By converting conventional flow cytometry beads into beads suitable for mass cytometry, our approach paves the way toward the broad implementation of bead-based assays in high-dimensional cell profiling studies by mass cytometry in biomedical research.