title: A universal chemical enrichment method for mapping the yeast N-glycoproteome by mass spectrometry (MS).
abstract: 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.
authors.affiliation: From the ‡School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
authors.forename: Weixuan
authors.initials: W
authors.lastname: Chen
authors.affiliation: From the ‡School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
authors.forename: Johanna M
authors.initials: JM
authors.lastname: Smeekens
authors.affiliation: From the ‡School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA Ronghu.Wu@chemistry.gatech.edu.
authors.forename: Ronghu
authors.initials: R
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D001897:Boronic Acids / Q000378:metabolism; D002853:Chromatography, Liquid; D006023:Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D006031:Glycosylation; D037102:Lectins / Q000378:metabolism; D013058:Mass Spectrometry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D012441:Saccharomyces cerevisiae
chemical_list: D001897:Boronic Acids; D006023:Glycoproteins; D037102:Lectins; D011134:Polysaccharides; D020543:Proteome; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase