Mirror-Cutting-Based Digestion Strategy Enables the In-Depth and Accuracy Characterization of N-Linked Protein Glycosylation
Mirror-Cutting-Based Digestion Strategy Enables the In-Depth and Accuracy Characterization of N-Linked Protein Glycosylation
复制标题
基于镜像切割的消化策略能够深入、准确地表征 N 连接蛋白糖基化
DOI：
10.1021/acs.jproteome.1c00333
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发表时间：
2021
影响因子：
4.4
通讯作者：
Ye Mingliang
中科院分区：
文献类型：
--
作者：
Chen Yao;Fang Zheng;Zhou Jiahua;Qin Hongqiang;Ye Mingliang
N-连接糖基化在多种生理和病理过程中起着重要作用，但由于糖蛋白消化不充分以及完整糖肽测定的离子碎片不完整，其分析范围仍然有限。本文提出了一种基于镜像切割的消化策略，该策略通过组合LysargiNase和胰蛋白酶的两种正交蛋白酶来表征蛋白质糖基化的宏观和微观异质性。使用上述两种蛋白酶，肽序列的B-或y-离子系列分别在MS/MS中增强，产生用于肽序列鉴定的互补光谱。超过27%（489/1778）的通过LysargiNase消化鉴定的位点特异性糖型未被胰蛋白酶消化覆盖，表明通过镜像切割方法提高了蛋白质序列和位点特异性糖型的覆盖率。在18 h MS分析中，总共从小鼠脑组织中鉴定了10，935个位点特异性糖型，这显著提高了蛋白质糖基化的覆盖率。有趣的是，用核心岩藻糖基化测定了27种甘露糖-6-磷酸（M6 P）糖型，人工检查后发现其中23种具有“Y-HexNAc-Fuc”离子。这是迄今为止首次报道的M6 P和岩藻糖基化的糖肽的共同修饰，其中的机制和功能仍需要进一步的探索。镜像切割消化策略在寻找其他复杂样品中缺失的糖蛋白方面也有很大的应用潜力，为糖生物学研究提供丰富的资源。
N-linked glycosylation plays important roles in multiple physiological and pathological processes, while the analysis coverage is still limited due to the insufficient digestion of glycoproteins, as well as incomplete ion fragments for intact glycopeptide determination. Herein, a mirror-cutting-based digestion strategy was proposed by combining two orthogonal proteases of LysargiNase and trypsin to characterize the macro- and micro-heterogeneity of protein glycosylation. Using the above two proteases, the b- or y-ion series of peptide sequences were, respectively, enhanced in MS/MS, generating the complementary spectra for peptide sequence identification. More than 27% (489/1778) of the site-specific glycoforms identified by LysargiNase digestion were not covered by trypsin digestion, suggesting the elevated coverage of protein sequences and site-specific glycoforms by the mirror-cutting method. Totally, 10,935 site-specific glycoforms were identified from mouse brain tissues in the 18 h MS analysis, which significantly enhanced the coverage of protein glycosylation. Intriguingly, 27 mannose-6-phosphate (M6P) glycoforms were determined with core fucosylation, and 23 of them were found with the “Y-HexNAc-Fuc” ions after manual checking. This is hitherto the first report of M6P and fucosylation co-modifications of glycopeptides, in which the mechanism and function still needs further exploration. The mirror-cutting digestion strategy also has great application potential in the exploration of missing glycoproteins from other complex samples to provide rich resources for glycobiology research.