Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.

Functional Attributes of Antibodies, Effector Cells, and Target Cells Affecting NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity.

Ab-dependent cellular cytotoxicity (ADCC) is one of the most important effector mechanisms of tumor-targeting Abs in current immunotherapies. In ADCC and other Ab-dependent activation of myeloid effector cells, close cell-cell contact (between effector and target cell) and formation of immunological synapses are required. However, we still lack basic knowledge on the principal factors influencing ADCC potential by therapeutic Abs. In this study we investigated the combined roles of five factors affecting human NK cell-mediated ADCC, namely: 1) Ag density, 2) target cell membrane composition, 3) IgG FcγR polymorphism, 4) FcγR-blocking cytophilic Abs, and 5) Ab fucosylation. We demonstrate that the magnitude of NK cell-mediated ADCC responses is predominantly influenced by Ag density and Ab fucosylation. Afucosylation consistently induced efficient ADCC, even at very low Ag density, where fucosylated target Abs did not elicit ADCC. On the side of the effector cell, the FcγRIIIa-Val/Phe158 polymorphism influenced ADCC potency, with NK cells expressing the Val158 variant showing more potent ADCC. In addition, we identified the sialic acid content of the target cell membrane as an important inhibitory factor for ADCC. Furthermore, we found that the presence and glycosylation status of aspecific endogenous Abs bound to NK cell FcγRIIIa (cytophilic Abs) determine the blocking effect on ADCC. These five parameters affect the potency of Abs in vitro and should be further tested as predictors of in vivo capacity.

Site-Specific Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of Individual Healthy Donors.

Fc gamma receptors (FcγRs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and FcγR impacts their interaction dramatically. Regarding FcγR glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map FcγRIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for FcγRIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying FcγRIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous FcγRIIIa on natural killer cells. This method will allow assessment of differences in FcγRIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions.

Ultraviolet Photodissociation of ESI- and MALDI-Generated Protein Ions on a Q-Exactive Mass Spectrometer.

The identification of molecular ions produced by MALDI or ESI strongly relies on their fragmentation to structurally informative fragments. The widely diffused fragmentation techniques for ESI multiply charged ions are either incompatible (ECD and ETD) or show lower efficiency (CID, HCD), with the predominantly singly charged peptide and protein ions formed by MALDI. In-source decay has been successfully adopted to sequence MALDI-generated ions, but it further increases spectral complexity, and it is not compatible with mass-spectrometry imaging. Excellent UVPD performances, in terms of number of fragment ions and sequence coverage, has been demonstrated for electrospray ionization for multiple proteomics applications. UVPD showed a much lower charge-state dependence, and so protein ions produced by MALDI may exhibit equal propensity to fragment. Here we report UVPD implementation on an Orbitrap Q-Exactive Plus mass spectrometer equipped with an ESI/EP-MALDI. UVPD of MALDI-generated ions was benchmarked against MALDI-ISD, MALDI-HCD, and ESI-UVPD. MALDI-UVPD outperformed MALDI-HCD and ISD, efficiently sequencing small proteins ions. Moreover, the singly charged nature of MALDI-UVPD avoids the bioinformatics challenges associated with highly congested ESI-UVPD mass spectra. Our results demonstrate the ability of UVPD to further improve tandem mass spectrometry capabilities for MALDI-generated protein ions. Data are available via ProteomeXchange with identifier PXD011526.

Mass Spectrometry Imaging, Laser Capture Microdissection, and LC-MS/MS of the Same Tissue Section.

Mass spectrometry imaging (MSI) is able to simultaneously record the distributions of hundreds of molecules directly from tissue. Rapid direct tissue analysis is essential for MSI in order to maintain spatial localization and acceptable measurement times. The absence of an explicit analyte separation/purification step means MSI lacks the depth of coverage of LC-MS/MS. In this work, we demonstrate how atmospheric pressure MALDI-MSI enables the same tissue section to be first analyzed by MSI, to identify regions of interest that exhibit distinct molecular signatures, followed by localized proteomics analysis using laser capture microdissection isolation and LC-MS/MS.

Design and Performance of a Novel Interface for Combined Matrix-Assisted Laser Desorption Ionization at Elevated Pressure and Electrospray Ionization with Orbitrap Mass Spectrometry.

Matrix-Assisted Laser Desorption Ionization, MALDI, has been increasingly used in a variety of biomedical applications, including tissue imaging of clinical tissue samples, and in drug discovery and development. These studies strongly depend on the performance of the analytical instrumentation and would drastically benefit from improved sensitivity, reproducibility, and mass/spatial resolution. In this work, we report on a novel combined MALDI/ESI interface, which was coupled to different Orbitrap mass spectrometers (Elite and Q Exactive Plus) and extensively characterized with peptide and protein standards, and in tissue imaging experiments. In our approach, MALDI is performed in the elevated pressure regime (5-8 Torr) at a spatial resolution of 15-30 µm, while ESI-generated ions are injected orthogonally to the interface axis. We have found that introduction of the MALDI-generated ions into an electrodynamic dual-funnel interface results in increased sensitivity characterized by a limit of detection of ∼400 zmol, while providing a mass measurement accuracy of 1 ppm and a mass resolving power of 120 000 in analysis of protein digests. In tissue imaging experiments, the MALDI/ESI interface has been employed in experiments with rat brain sections and was shown to be capable of visualizing and spatially characterizing very low abundance analytes separated only by 20 mDa. Comparison of imaging data has revealed excellent agreement between the MALDI and histological images.

Assessment of SRM, MRM(3) , and DIA for the targeted analysis of phosphorylation dynamics in non-small cell lung cancer.

Hypothesis-driven MS-based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data-independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high-throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well-informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain PTMs, as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM, and MRM cubed (MRM(3) ) in the analysis of phosphorylation dynamics throughout the phosphoinositide 3-kinase mechanistic target of rapamycin (PI3K-mTOR) and mitogen-activated protein kinase (MAPK) signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by postanalysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM(3) , allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites, gaining a so far unachieved in-depth view mTOR signaling events linked to tyrosine kinase inhibitor resistance in non-small cell lung cancer.

Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo.

Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 and H3K4 methylation from a proximal as well as a more distal site, but only if in a correct orientation. This plasticity indicates that the exact location of the attachment site, the native ubiquitin-lysine linkage and ubiquitination cycles are not critical for trans-histone crosstalk in vivo. The flexibility in crosstalk also indicates that other ubiquitination events may promote H3 methylation.

Single-step enrichment by Ti4+-IMAC and label-free quantitation enables in-depth monitoring of phosphorylation dynamics with high reproducibility and temporal resolution.

Quantitative phosphoproteomics workflows traditionally involve additional sample labeling and fractionation steps for accurate and in-depth analysis. Here we report a high-throughput, straightforward, and comprehensive label-free phosphoproteomics approach using the highly selective, reproducible, and sensitive Ti(4+)-IMAC phosphopeptide enrichment method. We demonstrate the applicability of this approach by monitoring the phosphoproteome dynamics of Jurkat T cells stimulated by prostaglandin E2 (PGE2) over six different time points, measuring in total 108 snapshots of the phosphoproteome. In total, we quantitatively monitored 12,799 unique phosphosites over all time points with very high quantitative reproducibility (average r > 0.9 over 100 measurements and a median cv < 0.2). PGE2 is known to increase cellular cAMP levels, thereby activating PKA. The in-depth analysis revealed temporal regulation of a wide variety of phosphosites associated not only with PKA, but also with a variety of other classes of kinases. Following PGE2 stimulation, several pathways became only transiently activated, revealing that in-depth dynamic profiling requires techniques with high temporal resolution. Moreover, the large publicly available dataset provides a valuable resource for downstream PGE2 signaling dynamics in T cells, and cAMP-mediated signaling in particular. More generally, our method enables in-depth, quantitative, high-throughput phosphoproteome screening on any system, requiring very little sample, sample preparation, and analysis time.

Phosphoproteome dynamics in onset and maintenance of oncogene-induced senescence.

Expression of the BRAF(V600E) oncoprotein is known to cause benign lesions, such as melanocytic nevi (moles). Despite the oncogenic function of mutant BRAF, these lesions are arrested by a cell-autonomous mechanism called oncogene-induced senescence. Infrequently, nevi can progress to malignant melanoma, through mechanisms that are incompletely understood. To gain more insight into this vital tumor-suppression mechanism, we performed a mass-spectrometry-based screening of the proteome and phosphoproteome in cycling and senescent cells and in cells with abrogated senescence. Proteome analysis of senescent cells revealed the up-regulation of established senescence biomarkers, including specific cytokines, but also several proteins not previously associated with senescence, including extracellular matrix-interacting. Using both general and targeted phosphopeptide enrichment by Ti(4+)-IMAC and phosphotyrosine antibody enrichment, we identified over 15,000 phosphorylation sites. Among the regulated phosphorylation sites we encountered components of the interleukin, BRAF/MAPK, and CDK-retinoblastoma pathways and several other factors. The extensive proteome and phosphoproteome dataset of BRAF(V600E)-expressing senescent cells provides molecular clues as to how oncogene-induced senescence is initiated, maintained, or evaded, serving as a comprehensive proteomic basis for functional validation.

Signal Transduction Reaction Monitoring Deciphers Site-Specific PI3K-mTOR/MAPK Pathway Dynamics in Oncogene-Induced Senescence.

We report a straightforward strategy to comprehensively monitor signal transduction pathway dynamics in mammalian systems. Combining targeted quantitative proteomics with highly selective phosphopeptide enrichment, we monitor, with great sensitivity, phosphorylation dynamics of the PI3K-mTOR and MAPK signaling networks. Our approach consists of a single enrichment step followed by a single targeted proteomics experiment, circumventing the need for labeling and immune purification while enabling analysis of selected phosphorylation nodes throughout signaling pathways. The need for such a comprehensive pathway analysis is illustrated by highlighting previously uncharacterized phosphorylation changes in oncogene-induced senescence, associated with diverse biological phenotypes and pharmacological intervention of the PI3K-mTOR pathway.

Spatio-temporal analysis of molecular determinants of neuronal degeneration in the aging mouse cerebellum.

The accumulation of cellular damage, including DNA damage, is hypothesized to contribute to aging-related neurodegenerative changes. DNA excision repair cross-complementing group 1 (Ercc1) knock-out mice represent an accepted model of neuronal aging, showing gradual neurodegenerative changes, including loss of synaptic contacts and cell body shrinkage. Here, we used the Purkinje cell-specific Ercc1 DNA-repair knock-out mouse model to study aging in the mouse cerebellum. We performed an in-depth quantitative proteomics analysis, using stable isotope dimethyl labeling, to decipher changes in protein expression between the early (8 weeks), intermediate (16 weeks), and late (26 weeks) stages of the phenotypically aging Ercc1 knock-out and healthy littermate control mice. The expression of over 5,200 proteins from the cerebellum was compared quantitatively, whereby 79 proteins (i.e. 1.5%) were found to be substantially regulated during aging. Nearly all of these molecular markers of the early aging onset belonged to a strongly interconnected network involved in excitatory synaptic signaling. Using immunohistological staining, we obtained temporal and spatial profiles of these markers confirming not only the proteomics data but in addition revealed how the change in protein expression correlates to synaptic changes in the cerebellum. In summary, this study provides a highly comprehensive spatial and temporal view of the dynamic changes in the cerebellum and Purkinje cell signaling in particular, indicating that synapse signaling is one of the first processes to be affected in this premature aging model, leading to neuron morphological changes, neuron degeneration, inflammation, and ultimately behavior disorders.

Predicting treatment outcome using kinome activity profiling in HER2+ breast cancer biopsies.

In this study, we measured the kinase activity profiles of 32 pre-treatment tumor biopsies of HER2-positive breast cancer patients. The aim of this study was to assess the prognostic potential of kinase activity levels, to identify potential mechanisms of resistance and to predict treatment success of HER2-targeted therapy combined with chemotherapy. Indeed, our system-wide kinase activity analysis allowed us to link kinase activity to treatment response. Overall, high kinase activity in the HER2-pathway was associated with good treatment outcome. We found eleven kinases differentially regulated between treatment outcome groups, including well-known players in therapy resistance, such as p38a, ERK, and FAK, and an unreported one, namely MARK1. Lastly, we defined an optimal signature of four kinases in a multiple logistic regression diagnostic test for prediction of treatment outcome (AUC = 0.926). This kinase signature showed high sensitivity and specificity, indicating its potential as predictive biomarker for treatment success of HER2-targeted therapy.

Innovative strategies for measuring kinase activity to accelerate the next wave of novel kinase inhibitors.

The development of protein kinase inhibitors (PKIs) has gained significance owing to their therapeutic potential for diseases like cancer. In addition, there has been a rise in refining kinase activity assays, each possessing unique biological and analytical characteristics crucial for PKI development. However, the PKI development pipeline experiences high attrition rates and approved PKIs exhibit unexploited potential because of variable patient responses. Enhancing PKI development efficiency involves addressing challenges related to understanding the PKI mechanism of action and employing biomarkers for precision medicine. Selecting appropriate kinase activity assays for these challenges can overcome these attrition rate issues. This review delves into the current obstacles in kinase inhibitor development and elucidates kinase activity assays that can provide solutions.

Progressive methylation of ageing histones by Dot1 functions as a timer.

Post-translational modifications of histone proteins have a crucial role in regulating gene expression. If efficiently re-established after chromosome duplication, histone modifications could help propagate gene expression patterns in dividing cells by epigenetic mechanisms. We used an integrated approach to investigate the dynamics of the conserved methylation of histone H3 Lys 79 (H3K79) by Dot1. Our results show that methylation of H3K79 progressively changes after histone deposition, which is incompatible with a rapid copy mechanism. Instead, methylation accumulates on ageing histones, providing the cell with a timer mechanism to directly couple cell-cycle length to changes in chromatin modification on the nucleosome core.

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available.

Novel kinome profiling technology reveals drug treatment is patient and 2D/3D model dependent in glioblastoma.

Glioblastoma is the deadliest brain cancer. One of the main reasons for poor outcome resides in therapy resistance, which adds additional challenges in finding an effective treatment. Small protein kinase inhibitors are molecules that have become widely studied for cancer treatments, including glioblastoma. However, none of these drugs have demonstrated a therapeutic activity or brought more benefit compared to the current standard procedure in clinical trials. Hence, understanding the reasons of the limited efficacy and drug resistance is valuable to develop more effective strategies toward the future. To gain novel insights into the method of action and drug resistance in glioblastoma, we established in parallel two patient-derived glioblastoma 2D and 3D organotypic multicellular spheroids models, and exposed them to a prolonged treatment of three weeks with temozolomide or either the two small protein kinase inhibitors enzastaurin and imatinib. We coupled the phenotypic evidence of cytotoxicity, proliferation, and migration to a novel kinase activity profiling platform (QuantaKinome™) that measured the activities of the intracellular network of kinases affected by the drug treatments. The results revealed a heterogeneous inter-patient phenotypic and molecular response to the different drugs. In general, small differences in kinase activation were observed, suggesting an intrinsic low influence of the drugs to the fundamental cellular processes like proliferation and migration. The pathway analysis indicated that many of the endogenously detected kinases were associated with the ErbB signaling pathway. We showed the intertumoral variability in drug responses, both in terms of efficacy and resistance, indicating the importance of pursuing a more personalized approach. In addition, we observed the influence derived from the application of 2D or 3D models in in vitro studies of kinases involved in the ErbB signaling pathway. We identified in one 3D sample a new resistance mechanism derived from imatinib treatment that results in a more invasive behavior. The present study applied a new approach to detect unique and specific drug effects associated with pathways in in vitro screening of compounds, to foster future drug development strategies for clinical research in glioblastoma.

Improving SRM assay development: a global comparison between triple quadrupole, ion trap, and higher energy CID peptide fragmentation spectra.

In proteomics, selected reaction monitoring (SRM) is rapidly gaining importance for targeted protein quantification. The triple quadrupole mass analyzers used in SRM assays allow for levels of specificity and sensitivity hard to accomplish by more standard shotgun proteomics experiments. Often, an SRM assay is built by in silico prediction of transitions and/or extraction of peptide precursor and fragment ions from a spectral library. Spectral libraries are typically generated from nonideal ion trap based shotgun proteomics experiments or synthetic peptide libraries, consuming considerable time and effort. Here, we investigate the usability of beam type CID (or "higher energy CID" (HCD)) peptide fragmentation spectra, as acquired using an Orbitrap Velos, to facilitate SRM assay development. Therefore, peptide fragmentation spectra, obtained by ion-trap CID, triple-quadrupole CID (QqQ-CID) and Orbitrap HCD, originating from digested cellular lysates, were compared. Spectral comparison and a dedicated correlation algorithm indicated significantly higher similarity between QqQ-CID and HCD fragmentation spectra than between QqQ-CID and ion trap-CID spectra. SRM transitions generated using a constructed HCD spectral library increased SRM assay sensitivity up to 2-fold, when compared to the use of a library created from more conventionally used ion trap-CID spectra, showing that HCD spectra can assist SRM assay development.

Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity.

Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.

In-Source Decay and Pseudo-MS3 of Peptide and Protein Ions Using Liquid AP-MALDI.

Atmospheric pressure MALDI on a Q-Exactive instrument was optimized for in-source decay and pseudo-MS3. The dependence of AP-MALDI ISD on the MALDI liquid matrix was investigated for peptides and proteins. The liquid matrices enabled long-life ISD signal, and exhibited high fragment ion yield and signal stability. Extensive a-, b-, c-, y-, and z-type fragment series were observed depending on the matrix used but were most extensive with 2,5-DHB. Complete sequence coverage of small peptide and intact protein-terminus sequence tags were obtained and confirmed using HCD as a pseudo-MS3 method. Graphical Abstract ᅟ.