Deciphering Protein <i>O</i>‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment

Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. <i>N</i>-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on <i>O</i>-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of <i>O</i>-glycans. Although <i>O</i>-glycosidase can catalyze the removal of core 1 and core 3 <i>O</i>-linked disaccharides from glycoproteins, analysis of other types of <i>O-</i>glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available <i>O-</i>protease and solid phase platform. This method enables the assignment of <i>O</i>-glycosylated peptides, <i>N</i>-glycan profile, sialyl <i>O</i>-glycopeptides linkage, and mapping of heterogeneous <i>O</i>-glycosylation. For the first time, we can analyze intact <i>O</i>-glycopeptides generated by <i>O</i>-protease and enriched using a solid-phase platform. We establish the method on standard glycoproteins, confirming known <i>O-</i>glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant <i>O</i>-glycosites and setting a basis set of <i>O</i>-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein <i>O</i>-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where <i>O</i>-glycosylation is central

Deciphering Protein <i>O</i>‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment

Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. <i>N</i>-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on <i>O</i>-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of <i>O</i>-glycans. Although <i>O</i>-glycosidase can catalyze the removal of core 1 and core 3 <i>O</i>-linked disaccharides from glycoproteins, analysis of other types of <i>O-</i>glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available <i>O-</i>protease and solid phase platform. This method enables the assignment of <i>O</i>-glycosylated peptides, <i>N</i>-glycan profile, sialyl <i>O</i>-glycopeptides linkage, and mapping of heterogeneous <i>O</i>-glycosylation. For the first time, we can analyze intact <i>O</i>-glycopeptides generated by <i>O</i>-protease and enriched using a solid-phase platform. We establish the method on standard glycoproteins, confirming known <i>O-</i>glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant <i>O</i>-glycosites and setting a basis set of <i>O</i>-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein <i>O</i>-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where <i>O</i>-glycosylation is central

Deciphering Protein <i>O</i>‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment

Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. <i>N</i>-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on <i>O</i>-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of <i>O</i>-glycans. Although <i>O</i>-glycosidase can catalyze the removal of core 1 and core 3 <i>O</i>-linked disaccharides from glycoproteins, analysis of other types of <i>O-</i>glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available <i>O-</i>protease and solid phase platform. This method enables the assignment of <i>O</i>-glycosylated peptides, <i>N</i>-glycan profile, sialyl <i>O</i>-glycopeptides linkage, and mapping of heterogeneous <i>O</i>-glycosylation. For the first time, we can analyze intact <i>O</i>-glycopeptides generated by <i>O</i>-protease and enriched using a solid-phase platform. We establish the method on standard glycoproteins, confirming known <i>O-</i>glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant <i>O</i>-glycosites and setting a basis set of <i>O</i>-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein <i>O</i>-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where <i>O</i>-glycosylation is central

Deciphering Protein <i>O</i>‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment

Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. <i>N</i>-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on <i>O</i>-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of <i>O</i>-glycans. Although <i>O</i>-glycosidase can catalyze the removal of core 1 and core 3 <i>O</i>-linked disaccharides from glycoproteins, analysis of other types of <i>O-</i>glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available <i>O-</i>protease and solid phase platform. This method enables the assignment of <i>O</i>-glycosylated peptides, <i>N</i>-glycan profile, sialyl <i>O</i>-glycopeptides linkage, and mapping of heterogeneous <i>O</i>-glycosylation. For the first time, we can analyze intact <i>O</i>-glycopeptides generated by <i>O</i>-protease and enriched using a solid-phase platform. We establish the method on standard glycoproteins, confirming known <i>O-</i>glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant <i>O</i>-glycosites and setting a basis set of <i>O</i>-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein <i>O</i>-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where <i>O</i>-glycosylation is central

Deciphering Protein <i>O</i>‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment

Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. <i>N</i>-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on <i>O</i>-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of <i>O</i>-glycans. Although <i>O</i>-glycosidase can catalyze the removal of core 1 and core 3 <i>O</i>-linked disaccharides from glycoproteins, analysis of other types of <i>O-</i>glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available <i>O-</i>protease and solid phase platform. This method enables the assignment of <i>O</i>-glycosylated peptides, <i>N</i>-glycan profile, sialyl <i>O</i>-glycopeptides linkage, and mapping of heterogeneous <i>O</i>-glycosylation. For the first time, we can analyze intact <i>O</i>-glycopeptides generated by <i>O</i>-protease and enriched using a solid-phase platform. We establish the method on standard glycoproteins, confirming known <i>O-</i>glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant <i>O</i>-glycosites and setting a basis set of <i>O</i>-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein <i>O</i>-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where <i>O</i>-glycosylation is central

Deciphering Protein <i>O</i>‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment

Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. <i>N</i>-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on <i>O</i>-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of <i>O</i>-glycans. Although <i>O</i>-glycosidase can catalyze the removal of core 1 and core 3 <i>O</i>-linked disaccharides from glycoproteins, analysis of other types of <i>O-</i>glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available <i>O-</i>protease and solid phase platform. This method enables the assignment of <i>O</i>-glycosylated peptides, <i>N</i>-glycan profile, sialyl <i>O</i>-glycopeptides linkage, and mapping of heterogeneous <i>O</i>-glycosylation. For the first time, we can analyze intact <i>O</i>-glycopeptides generated by <i>O</i>-protease and enriched using a solid-phase platform. We establish the method on standard glycoproteins, confirming known <i>O-</i>glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant <i>O</i>-glycosites and setting a basis set of <i>O</i>-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein <i>O</i>-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where <i>O</i>-glycosylation is central