O-glycosylation is a highly diverse and complex form of protein post-translational modification. Mucin-type O-glycosylation is initiated by the transfer of N-acetyl-galactosamine (GalNAc) to the hydroxyl group of serine, threonine and tyrosine residues through catalysis by a family of glycosyltransferases, the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (E.C. 2.4.1.41) that are conserved across metazoans. In the last decade, structural characterization of glycosylation has substantially advanced due to the development of analytical methods and advances in mass spectrometry. However, O-glycosite mapping remains challenging since mucin-type O-glycans are densely packed, often protecting proteins from cleavage by proteases. Adding to the complexity is the fact that a given glycosite can be modified by different glycans, resulting in an array of glycoforms rising from one glycosite. In this study, we investigated conditions of solid phase extraction (SPE) enrichment, protease digestion, and Electron-transfer/Higher Energy Collision Dissociation (EThcD) fragmentation to optimize identification of O-glycosites in densely glycosylated proteins. Our results revealed that anion-exchange stationary phase is sufficient for glycopeptide enrichment; however, the use of a hydrophobic-containing sorbent was detrimental to the binding of polar-hydrophilic glycopeptides. Different proteases can be employed for enhancing coverage of O-glycosites, while derivatization of negatively charged amino acids or sialic acids would enhance the identification of a short O-glycopeptides. Using a longer than normal electron transfer dissociation (ETD) reaction time, we obtained enhanced coverage of peptide bonds that facilitated the localization of O-glycosites.

O-糖基化是一种高度多样化和复杂的蛋白质翻译后修饰形式。粘蛋白型 O-糖基化由 N-乙酰半乳糖胺 (GalNAc) 通过糖基转移酶家族 UDP-GalNAc: 多肽 N-乙酰半乳糖胺基转移酶 (EC 2.4.1.41）在后生动物中是保守的。在过去十年中，由于分析方法的发展和质谱技术的进步，糖基化的结构表征有了很大的进步。然而，O-糖苷位图谱仍然具有挑战性，因为粘蛋白型 O-聚糖是密集堆积的，通常可以保护蛋白质免受蛋白酶的切割。更复杂的是，给定的糖位点可以被不同的聚糖修饰，导致从一个糖位点升起的一系列糖型。在本研究中，我们研究了固相萃取 (SPE) 富集、蛋白酶消化和电子转移/高能碰撞解离 (ETHcD) 碎裂的条件，以优化密集糖基化蛋白质中 O-糖苷的鉴定。我们的结果表明，阴离子交换固定相足以富集糖肽。然而，使用含有疏水性的吸附剂不利于极性亲水性糖肽的结合。可以使用不同的蛋白酶来增强 O-糖苷的覆盖率，而带负电荷的氨基酸或唾液酸的衍生化将增强对短 O-糖肽的鉴定。使用比正常更长的电子转移解离 (ETD) 反应时间，