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Oral immunotherapy (OIT) leads to suppression of mast cell and basophil degranulation along with changes in the adaptive immune response.This study aimed to determine how rapidly these effects occur during OIT and more broadly, the kinetics of basophil and mast cell suppression throughout the course of therapy.Twenty participants, age 4 to 12 years, were enrolled in a peanut OIT trial and assessed for desensitization and sustained unresponsiveness after 9 months of therapy. Blood was collected 5 times in the first month and then intermittently throughout to quantify immunoglobulins and assess basophil activation by CD63, CD203c, and phosphorylated SYK (pSYK).Twelve of 16 participants that completed the trial were desensitized after OIT, with 9 achieving sustained unresponsiveness after discontinuing OIT for 4 weeks. Basophil hyporesponsiveness, defined by lower CD63 expression, was detected as early as day 90. pSYK was correlated with CD63 expression, and there was a significant decrease in pSYK by day 250. CD203c expression remained unchanged throughout therapy. Interestingly, although basophil activation was decreased across the cohort during OIT, basophil activation did not correlate with individual clinical outcomes. Serum peanut-specific IgGSuppression of basophil activation occurs within the first 90 days of peanut OIT, ultimately leading to suppression of signaling through pSYK.

BACKGROUND: Circulating IgE and subsequent severe allergic reactions to peanut are sustained and propagated by recall of peanut allergen-specific memory B cells. OBJECTIVES: This study aimed to determine whether targeting mouse and human CD22 on peanut-specific memory B cells induces tolerance to peanut allergens. METHODS: Siglec-engaging tolerance-inducing antigenic liposomes (STALs) codisplaying peanut allergens (Ara h 1, Ara h 2, or Ara h 3) and high-affinity CD22 ligand (CD22L-STALs) were employed in various mouse models (BALB/cJ, C57BL/6, human CD22 transgenic, and NSG) of peanut allergy. To investigate memory B cells, a conferred memory model was used in which splenocytes from peanut-sensitized mice were transferred into naive animals. Reconstituted mice received either CD22L-STALs or an immunogenic liposome control, followed by a peanut allergen boost and later a challenge with individual peanut allergens. To assess the effects of CD22L-STALs on human B cells, PBMCs were injected into NSG mice, followed by administration of human CD22L-STALs (hCD22L-STALs) and later a whole peanut extract boost. Blood was collected to quantify WPE- and Ara h 1–, 2–, and 3–specific immunoglobulins. RESULTS: Mouse CD22L-STALs (mCD22L-STALs) significantly suppressed systemic memory to Ara h 1, Ara h 2, and Ara h 3 in BALB/cJ and C57BL/6 mice, as demonstrated by reduced allergen-specific IgE, IgG(1), and anaphylaxis on challenge. Importantly, 2 doses of mCD22L-STALs led to prolonged tolerance for at least 3 months. hCD22L-STALs displayed similar suppression in mice expressing human CD22 on B cells. Finally, human B cells were tolerized in vivo in NSG mice by hCD22L-STALs. CONCLUSIONS: Antigen-specific exploitation of CD22 on memory B cells can induce systemic immune tolerance.

Antigen-specific immunoglobulin responses have yet to be studied at the oral mucosal surface during peanut oral immunotherapy (PnOIT).We aimed to quantify salivary peanut-specific IgG4 (PNsIgG4) and IgA (PNsIgA) and total IgG4 and IgA in participants from the Immune Tolerance Network's IMPACT study, a phase 2 PnOIT trial.Peanut-allergic children, aged 12 months to younger than 48 months at screening, were enrolled and randomized to PnOIT or placebo oral immunotherapy (OIT) for 134 weeks. Per-protocol analysis included 69 PnOIT and 23 placebo participants. Double-blind, placebo-controlled food challenges were conducted at weeks 134 and 160 to assess desensitization and remission, respectively. Saliva samples were collected at baseline and 30, 82, 134, and 160 weeks to quantify PNsIgG4, PNsIgA, and total IgG4 and IgA.Participants who received PnOIT experienced significant increases in PNsIgG4 in saliva, whereas participants on placebo did not (P.01 at all time points). The PNsIgA/total IgA ratio was also significantly increased in participants treated with PnOIT when compared with those receiving placebo at 30 and 82 weeks (P.05). During PnOIT, desensitized participants had increased PNsIgA that plateaued, whereas the not desensitized/no remission group did not change over time. Interestingly, when the PnOIT group was evaluated by clinical outcome, PNsIgA was higher at baseline in the not desensitized/no remission group than in the desensitized/remission group (P.05).PnOIT induces substantial increases in allergen-specific IgG4 and IgA in saliva. These data provide insight into OIT-induced mucosal responses and suggest the utility of these easily obtained samples for biomarker development.

Proteins are continuously synthesized during cell growth and proliferation. At the same time, excessive and misfolded proteins have to be degraded, otherwise they are a burden to cells. Protein degradation is essential to maintain proteostasis in cells, and dysfunction of protein degradation systems results in numerous diseases such as cancer and neurodegenerative diseases. Despite the importance of protein degradation, the degradation pathways of many proteins remain to be explored. Here, we comprehensively investigated the degradation of newly synthesized proteins in human cells by integrating metabolic labeling, click chemistry, and multiplexed proteomics, and systematic and quantitative analysis of newly synthesized proteins first revealed the degradation pathways of many proteins. Bioinformatic analysis demonstrates that proteins degraded through two major pathways have distinct properties and functions. Proteins degraded through the ubiquitin-proteasome pathway contain more disordered structures, whereas those through the autophagy-lysosome pathway have significantly higher hydrophobicity. Systematic and quantitative investigation of the dynamics of newly synthesized proteins provides unprecedented and valuable information about protein degradation, which leads to a better understanding of protein properties and cellular activities., Systematic quantification of the dynamics of newly synthesized proteins first reveals the degradation pathways of many proteins in human cells, and proteins degraded through each of the two major pathways have distinct properties and functions.

Protein glycosylation is ubiquitous in biological systems and essential for cell survival. However, the heterogeneity of glycans and the low abundance of many glycoproteins complicate their global analysis. Chemical methods based on reversible covalent interactions between boronic acid and glycans have great potential to enrich glycopeptides, but the binding affinity is typically not strong enough to capture low-abundance species. Here, we develop a strategy using dendrimer-conjugated benzoboroxole to enhance the glycopeptide enrichment. We test the performance of several boronic acid derivatives, showing that benzoboroxole markedly increases glycopeptide coverage from human cell lysates. The enrichment is further improved by conjugating benzoboroxole to a dendrimer, which enables synergistic benzoboroxole–glycan interactions. This robust and simple method is highly effective for sensitive glycoproteomics analysis, especially capturing low-abundance glycopeptides. Importantly, the enriched glycopeptides remain intact, making the current method compatible with mass-spectrometry-based approaches to identify glycosylation sites and glycan structures., Understanding the functions of protein glycosylation critically depends on methods to efficiently enrich glycoproteins from complex samples. Here, the authors develop a strategy using dendrimer-conjugated benzoboroxole to enhance glycopeptide enrichment, providing the basis for more comprehensive glycoprotein analyses.

Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, the reaction incompletion and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with the commonly used reagents, i.e., dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, or tris(3-hydroxypropyl)phosphine, and alkylation with iodoacetamide, acrylamide, N-ethylmaleimide, or 4-vinylpyridine. By using digested peptides from a yeast whole-cell lysate, the number of proteins and peptides identified were very similar using four different reducing reagents. The results from four alkylating reagents, however, were dramatically different with iodoacetamide giving the highest number of peptides with alkylated cysteine and the lowest number of peptides with incomplete cysteine alkylation and side reactions. Alkylation conditions with iodoacetamide were further optimized. To identify more peptides with cysteine, Thiopropyl-Sepharose 6B resins were used to enrich them, and the optimal conditions were employed for the reduction and alkylation. The enrichment resulted in over three times more cysteine-containing peptides than without enrichment. Systematic evaluation of the reduction and alkylation with different reagents can aid in a better design of bottom-up proteomic experiments.

Glycoproteins on the cell surface are ubiquitous and essential for cells to interact with the extracellular matrix, communicate with other cells, and respond to environmental cues. Although surface sialoglycoproteins can dramatically impact cell properties and represent different cellular statuses, global and site-specific analysis of sialoglycoproteins only on the cell surface is extraordinarily challenging. An effective method integrating metabolic labeling, copper-free click chemistry and mass spectrometry-based proteomics was developed to globally and site-specifically analyze surface N-sialoglycoproteins. Surface sialoglycoproteins metabolically labeled with a functional group were specifically tagged through copper-free click chemistry, which is ideal because it is quick, specific and occurs under physiological conditions. Sequentially tagged sialoglycoproteins were enriched for site-specific identification by mass spectrometry. Systematic and quantitative analysis of the surface N-sialoglycoproteome in cancer cells with distinctive invasiveness demonstrated many N-sialoglycoproteins up-regulated in invasive cells, the majority of which contained cell adhesion-related domains. This method is very effective to globally and site-specifically analyze N-sialoglycoproteins on the cell surface, and will have extensive applications in the biological and biomedical research communities. Site-specific information regarding surface sialoglycoproteins can serve as biomarkers for disease detection, targets for vaccine development and drug treatment.

Tissue engineering strategies involving the in vivo delivery of recombinant growth factors are often limited by the inability of biomaterials to spatially control diffusion of the delivered protein within the site of interest. The poor spatiotemporal control provided by porous collagen sponges, which are used for the clinical delivery of bone morphogenetic protein-2 (BMP-2) for bone regeneration, has necessitated the use of supraphysiological protein doses, leading to inflammation and heterotopic ossification. This study describes a novel tissue engineering strategy to spatially control rapid BMP-2 diffusion from collagen sponges in vivo by creating a high-affinity BMP-2 sink around the collagen sponge. We designed an electrospun poly-ɛ-caprolactone nanofiber mesh containing physically entrapped heparin microparticles, which have been previously demonstrated to bind and retain large amounts of BMP-2. Nanofiber meshes containing 0.05 and 0.10 mg of microparticles/cm2 demonstrated increased BMP-2 binding and decreased BMP-2 release in vitro compared with meshes without microparticles. However, when microparticle-containing meshes were used in vivo to limit the diffusion of BMP-2 delivered by using collagen sponges in a rat femoral defect, no differences in heterotopic ossification or biomechanical properties were observed. Further investigation revealed that, although BMP-2 binding to heparin microparticles was rapid, the presence of serum components attenuated microparticle-BMP-2 binding and increased BMP-2 release in vitro. These observations provide a plausible explanation for the results observed in vivo and suggest that competitive protein binding in vivo may hinder the ability of affinity-based biomaterials to modulate growth factor delivery.

Glycosylation is one of the most common and important protein modifications in biological systems. Many glycoproteins naturally occur at low abundances, which makes comprehensive analysis extremely difficult. Additionally, glycans are highly heterogeneous, which further complicates analysis in complex samples. Lectin enrichment has been commonly used, but each lectin is inherently specific to one or several carbohydrates, and thus no single or collection of lectin(s) can bind to all glycans. Here we have employed a boronic acid-based chemical method to universally enrich glycopeptides. The reaction between boronic acids and sugars has been extensively investigated, and it is well known that the interaction between boronic acid and diols is one of the strongest reversible covalent bond interactions in an aqueous environment. This strong covalent interaction provides a great opportunity to catch glycopeptides and glycoproteins by boronic acid, whereas the reversible property allows their release without side effects. More importantly, the boronic acid-diol recognition is universal, which provides great capability and potential for comprehensively mapping glycosylation sites in complex biological samples. By combining boronic acid enrichment with PNGase F treatment in heavy-oxygen water and MS, we have identified 816 N-glycosylation sites in 332 yeast proteins, among which 675 sites were well-localized with greater than 99% confidence. The results demonstrated that the boronic acid-based chemical method can effectively enrich glycopeptides for comprehensive analysis of protein glycosylation. A general trend seen within the large data set was that there were fewer glycosylation sites toward the C termini of proteins. Of the 332 glycoproteins identified in yeast, 194 were membrane proteins. Many proteins get glycosylated in the high-mannose N-glycan biosynthetic and GPI anchor biosynthetic pathways. Compared with lectin enrichment, the current method is more cost-efficient, generic, and effective. This method can be extensively applied to different complex samples for the comprehensive analysis of protein glycosylation.

Protein secretion is essential for numerous cellular activities, and secreted proteins in bodily fluids are a promising and noninvasive source of biomarkers for disease detection. Systematic analysis of secreted proteins and glycoproteins will provide insight into protein function and cellular activities. Yeast (Saccharomyces cerevisiae) is an excellent model system for eukaryotic cells, but global analysis of secreted proteins and glycoproteins in yeast is challenging due to the low abundances of secreted proteins and contamination from high-abundance intracellular proteins. Here, by using mild separation of secreted proteins from cells, we comprehensively identified and quantified secreted proteins and glycoproteins through inhibition of glycosylation and mass spectrometry-based proteomics. In biological triplicate experiments, 245 secreted proteins were identified, and comparison with previous experimental and computational results demonstrated that many identified proteins were located in the extracellular space. Most quantified secreted proteins were down-regulated from cells treated with an N-glycosylation inhibitor (tunicamycin). The quantitative results strongly suggest that the secretion of these down-regulated proteins was regulated by glycosylation, while the secretion of proteins with minimal abundance changes was contrarily irrelevant to protein glycosylation, likely being secreted through nonclassical pathways. Glycoproteins in the yeast secretome were globally analyzed for the first time. A total of 27 proteins were quantified in at least two protein and glycosylation triplicate experiments, and all except one were down-regulated under N-glycosylation inhibition, which is solid experimental evidence to further demonstrate that the secretion of these proteins is regulated by their glycosylation. These results provide valuable insight into protein secretion, which will further advance protein secretion and disease studies.

Divergence between prokaryotic and eukaryotic ribosomal RNA (rRNA) and among eukaryotic ribosomal RNAs is focused in expansion segments (ESs). Eukaryotic ribosomes are significantly larger than prokaryotic ribosomes partly because of their ESs. We hypothesize that larger rRNAs of complex organisms could confer increased functionality to the ribosome. Here, we characterize the binding partners of Saccharomyces cerevisiae expansion segment 7 (ES7), which is the largest and most variable ES of the eukaryotic large ribosomal subunit and is located at the surface of the ribosome. In vitro RNA-protein pull-down experiments using ES7 as a bait indicate that ES7 is a binding hub for a variety of non-ribosomal proteins essential to ribosomal function in eukaryotes. ES7-associated proteins observed here cluster into four groups based on biological process, (i) response to abiotic stimulus (e.g., response to external changes in temperature, pH, oxygen level, etc.), (ii) ribosomal large subunit biogenesis, (iii) protein transport and localization, and (iv) transcription elongation. Seven synthetases, Ala-, Arg-, Asp-, Asn-, Leu-, Lys- and TyrRS, appear to associate with ES7. Affinities of AspRS, TyrRS and LysRS for ES7 were confirmed by in vitro binding assays. The results suggest that ES7 in S. cerevisiae could play a role analogous to the multi-synthetase complex present in higher order organisms and could be important for the appropriate function of the ribosome. Thermal denaturation studies and footprinting experiments confirm that isolated ES7 is stable and maintains a near-native secondary and tertiary structure.

Protein dynamics are essential in regulating nearly every cellular event, and aberrant proteostasis is the source of many diseases. It is extraordinarily difficult to globally study protein dynamics and accurately measure their half-lives. Here we have developed a chemical proteomics method integrating protein labeling, click chemistry and multiplexed proteomics, which overcomes current challenges with existing methods. Labeling with both azidohomoalanine (AHA) and heavy lysine allows us to selectively enrich newly synthesized proteins, clearly distinguish them from existing proteins, and reduce the impact of heavy amino acid recycling. Moreover, multiplexed proteomics enables us to quantify proteins at multiple time points simultaneously, thus increasing the accuracy of measuring protein abundance changes and their half-lives. Systematic investigation of newly synthesized protein dynamics will provide insight into proteostasis and the molecular mechanisms of disease.

For decades, statins have been widely used to lower cholesterol levels by inhibiting the enzyme HMG Co-A reductase (HMGCR). It is well-known that statins have pleiotropic effects including improving endothelial function and inhibiting vascular inflammation and oxidation. However, the cellular responses to statins and corresponding pleiotropic effects are largely unknown at the proteome level. Emerging mass spectrometry-based proteomics provides a unique opportunity to systemically investigate protein and phosphoprotein abundance changes as a result of statin treatment. Many lipid-related protein abundances were increased in HepG2 cells treated by atorvastatin, including HMGCR, FDFT, SQLE, and LDLR, while the abundances of proteins involved in cellular response to stress and apoptosis were decreased. Comprehensive analysis of protein phosphorylation demonstrated that several basic motifs were enriched among down-regulated phosphorylation sites, which indicates that kinases with preference for these motifs, such as protein kinase A and protein kinase C, have attenuated activities. Phosphopeptides on a group of G-protein modulators were up-regulated, which strongly suggests that cell signal rewiring was a result of the effect of protein lipidation by the statin. This work provides a global view of liver cell responses to atorvastatin at the proteome and phosphoproteome levels, which provides insight into the pleiotropic effects of statins.

Cell surface N-glycoproteins play extraordinarily important roles in cell–cell communication, cell–matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide-N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 N-glycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies.