Differential Quantitative Determination of Site-Specific Intact N-Glycopeptides in Serum Haptoglobin between Hepatocellular Carcinoma and Cirrhosis Using LC-EThcD-MS/MS.,Journal of Proteome Research

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Differential Quantitative Determination of Site-Specific Intact N-Glycopeptides in Serum Haptoglobin between Hepatocellular Carcinoma and Cirrhosis Using LC-EThcD-MS/MS.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2018-10-29 , DOI: 10.1021/acs.jproteome.8b00654
Jianhui Zhu ₁ , Zhengwei Chen ₂ , Jie Zhang ₁ , Mingrui An ₁ , Jing Wu ₁ , Qing Yu ₃ , St John Skilton ₄ , Marshall Bern ₄ , K Ilker Sen ₄ , Lingjun Li _{2,

3} , David M Lubman ₁

Affiliation

Intact N-glycopeptide analysis remains challenging due to the complexity of glycopeptide structures, low abundance of glycopeptides in protein digests, and difficulties in data interpretation/quantitation. Herein, we developed a workflow that involved advanced methodologies, the EThcD-MS/MS fragmentation method and data interpretation software, for differential analysis of the microheterogeneity of site-specific intact N-glycopeptides of serum haptoglobin between early hepatocellular carcinoma (HCC) and liver cirrhosis. Haptoglobin was immunopurified from 20 μL of serum in patients with early HCC, liver cirrhosis, and healthy controls, respectively, followed by trypsin/GluC digestion, glycopeptide enrichment, and LC-EThcD-MS/MS analysis. Identification and differential quantitation of site-specific N-glycopeptides were performed using a combination of Byonic and Byologic software. In total, 93, 87, and 68 site-specific N-glycopeptides were identified in early HCC, liver cirrhosis, and healthy controls, respectively, with high confidence. The increased variety of N-glycopeptides in liver diseases compared to healthy controls was due to increased branching with hyper-fucosylation and sialylation. Differential quantitation analysis showed that 5 site-specific N-glycopeptides on sites N184 and N241 were significantly elevated in early HCC compared to cirrhosis ( p < 0.05) and normal controls ( p ≤ 0.001). The result demonstrates that the workflow provides a strategy for detailed profiles of N-glycopeptides of patient samples as well as for relative quantitation to determine the level changes in site-specific N-glycopeptides between disease states.

中文翻译：

使用 LC-ETHcD-MS/MS 差异定量测定肝细胞癌和肝硬化血清触珠蛋白中位点特异性完整 N-糖肽。

由于糖肽结构的复杂性、蛋白质消化物中糖肽的丰度低以及数据解释/定量的困难，完整的 N-糖肽分析仍然具有挑战性。在此，我们开发了一个涉及先进方法、EThcD-MS/MS 裂解方法和数据解释软件的工作流程，用于早期肝细胞癌 (HCC) 和肝癌之间血清触珠蛋白位点特异性完整 N-糖肽的微观异质性差异分析肝硬化。分别从早期 HCC 患者、肝硬化患者和健康对照者的 20 μL 血清中免疫纯化触珠蛋白，然后进行胰蛋白酶/GluC 消化、糖肽富集和 LC-EThcD-MS/MS 分析。使用 Byonic 和 Byologic 软件的组合进行位点特异性 N-糖肽的鉴定和差异定量。总共，在早期 HCC、肝硬化和健康对照中分别鉴定出 93、87 和 68 个位点特异性 N-糖肽，且可信度较高。与健康对照相比，肝脏疾病中 N-糖肽种类的增加是由于高岩藻糖基化和唾液酸化的分支增加所致。差异定量分析显示，与肝硬化 (p < 0.05) 和正常对照 (p ≤ 0.001) 相比，早期 HCC 中 N184 和 N241 位点上的 5 个位点特异性 N-糖肽显着升高。结果表明，该工作流程提供了一种策略，用于详细分析患者样本的 N-糖肽以及相对定量以确定疾病状态之间位点特异性 N-糖肽的水平变化。

更新日期：2018-10-30

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