Comprehensive Analysis of Protein N‑Glycosylation Sites by Combining Chemical Deglycosylation with LC–MS

Glycosylation is one of the most important protein modifications in biological systems. It plays a critical role in protein folding, trafficking, and stability as well as cellular events such as immune response and cell-to-cell communication. Aberrant protein glycosylation is correlated with several diseases including diabetes, cancer, and infectious diseases. The heterogeneity of glycans makes comprehensive identification of protein glycosylation sites very difficult by MS because it is challenging to match mass spectra to peptides that contain different types of unknown glycans. We combined a chemical deglycosylation method with LC–MS-based proteomics techniques to comprehensively identify protein N-glycosylation sites in yeast. On the basis of the differences in chemical properties between the amide bond of the N-linkage and the glycosidic bond of the O-linkage of sugars, O-linked sugars were removed and only the innermost N-linked GlcNAc remained, which served as a mass tag for MS analysis. This chemical deglycosylation method allowed for the identification of 555 protein N-glycosylation sites in yeast by LC–MS, which is 46% more than those obtained from the parallel experiments using the Endo H cleavage method. A total of 250 glycoproteins were identified, including 184 membrane proteins. This method can be extensively used for other biological samples

Image1_Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice.tif

BackgroundEarly dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains.ObjectiveWe aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains.MethodsC57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed.ResultsWithout oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge.ConclusionAllergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate.</p

Table2_Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice.docx

BackgroundEarly dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains.ObjectiveWe aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains.MethodsC57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed.ResultsWithout oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge.ConclusionAllergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate.</p

Datasheet1_Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice.docx

BackgroundEarly dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains.ObjectiveWe aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains.MethodsC57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed.ResultsWithout oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge.ConclusionAllergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate.</p

Table1_Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice.docx

BackgroundEarly dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains.ObjectiveWe aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains.MethodsC57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed.ResultsWithout oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge.ConclusionAllergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate.</p