Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry
Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry
复制标题
通过 EThcD 和 HCD 质谱法对人血清糖蛋白组中的单和多 O-糖基化肽进行系统分析
DOI：
10.1016/j.jprot.2017.09.014
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发表时间：
2018-01-06
影响因子：
3.3
通讯作者：
Qian, Xiaohong
中科院分区：
文献类型：
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作者：
Zhang, Yong;Xie, Xinfang;Qian, Xiaohong
人类血清已被广泛研究，以通过全球蛋白质组分析来识别生物标志物。改变的O-糖蛋白组与人类的病理状态有关，包括癌症、炎症性疾病和退行性疾病，是疾病生物标志物的一个有吸引力的来源。由于O-糖基化的微观异质性和宏观异质性，对人血清中O-糖基化的定位分析仍然具有挑战性。在这里，我们开发了一种系统的策略，将多酶消化、多维分离用于样品制备和高分辨率串联MS与Byonic软件相结合用于完整的O-糖肽的表征。结果表明，多酶消化或多维分离可以提高样品制备的效率，而且EThcD不仅适用于单一O-糖基化多肽(50.3%)的鉴定，而且适用于双重(21.2%)和三重(28.5%)O-糖基化多肽的鉴定。在严格的评分标准下，从人血清中总共鉴定出499个非冗余的完整O-糖肽、173个O-糖基化位点和6种类型的O-糖链，它们来自49个O-糖蛋白基团，其中121个新的O-糖基化位点。目前，这是从人类血清样本中获得的最大的位点特异性天然O-糖蛋白组数据集。我们期望这项研究开发的策略将有助于深入分析人类血清中天然的O-糖蛋白组，并为了解蛋白质O-糖基化在人类健康和疾病中的功能作用提供机会。生物学意义：改变的O-糖蛋白组与人类的病理状态有关，是疾病生物标志物的一个有吸引力的来源。然而，由于O-糖基化的微观异质性(不同的糖形式附着在一个糖基化位点上)和宏观的异质性(位点占有率)，特定位点的O-糖基化分析是具有挑战性的。这项研究利用了新的裂解方法EThcD的固有特性，与碰撞诱导解离(HCD)方法相比，EThcD方法提供了更完整的O-糖基化多肽的裂解信息和更确定的O-糖链的位置定位。结果表明，多酶消化或多维分离可以提高样品制备的效率，EThcD不仅适用于单一O-糖基化多肽(50.3%)的鉴定，而且适用于双重O-糖基化多肽(21.2%)和三重O-糖基化多肽(28.5%)的鉴定。最后，我们从人血清样本中获得了一个最大的位点特异性天然O-糖蛋白组数据集。此外，从IgA肾病(IgAN)患者和健康献血者的血清样本中对完整的O-糖肽进行了定量分析，结果表明该策略具有发现O-糖基化生物标志物的潜力。我们期望本研究开发的策略将促进对人血清中天然O-糖蛋白组的深入分析，并为了解蛋白质O-糖基化在人类健康和疾病中的功能作用提供令人兴奋的机会。
Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O-glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macro heterogeneity of O-glycosylation, site-specific O-glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) 0-glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O-glycopeptides, 173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human serum, including 121 novel O-glycosylation sites. Currently, this is the largest data set of site-specific native O-glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases.Biological significance: The altered O-glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O-glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O-glycosylation.In this work, we developed a systematic strategy for intact O-glycopeptide characterization. This study took advantage of the inherent properties of the new fragmentation method called EThcD, which provides more complete fragmentation information about O-glycosylated peptides and a more confident site localization of O-glycans than collision-induced dissociation (HCD). We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD was not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Finally, we got a largest data set of site-specific native O-glycoproteome from human serum samples. Furthermore, quantitative analysis of intact O-glycopeptides from the serum samples of IgA nephropathy (IgAN) patients and healthy donors was performed, and the results showed the potential of the strategy to discover O-glycosylation biomarkers.We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and lead to exciting opportunities to understand the functional roles of protein O-glycosylation in human health and diseases.