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Abstract：
Mucin-domain glycoproteins are characterized by their high density of glycosylated serine and threonine residues, which complicates their analysis by mass spectrometry. The dense glycosylation renders the protein backbone inaccessible to workhorse proteases like trypsin, the vast heterogeneity of glycosylation often results in ion suppression from unmodified peptides, and search algorithms struggle to confidently analyze and site-localize O-glycosites. We have made a number of advances to address these challenges, rendering mucinomics possible for the first time. Here, we summarize these contributions and provide a detailed protocol for mass spectrometric analysis of mucin-domain glycoproteins. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Enrichment of mucin-domain glycoproteins Basic Protocol 2: Enzymatic digestion of mucin-domain glycoprotein(s) Basic Protocol 3: Mass spectrometry data collection for O-glycopeptides Basic Protocol 4: Mass spectrometry data analysis of O-glycopeptides.
摘要：
粘蛋白结构域糖蛋白的特征在于其高密度的糖基化丝氨酸和苏氨酸残基,这使得他们的质谱分析变得复杂。密集的糖基化使得蛋白质骨架无法接触到像胰蛋白酶这样的主力蛋白酶，糖基化的巨大异质性通常会导致未修饰肽的离子抑制，搜索算法难以自信地分析和定位O-糖位点。我们在应对这些挑战方面取得了一些进展,第一次使粘液组学成为可能。这里,我们总结了这些贡献，并提供了粘蛋白结构域糖蛋白质谱分析的详细方案。©2024Wiley期刊有限责任公司。基本方案1:粘蛋白结构域糖蛋白的富集基本方案2:粘蛋白结构域糖蛋白的酶消化基本方案3:O-糖肽的质谱数据收集基本方案4:O-糖肽的质谱数据分析。
