Zeng Wenjuan, Zheng Shanshan, Su Tao, Cheng Jiahan, Mao Yonghong, Zhong Yi, Liu Yueqiu, Chen Jianhai, Zhao Wanjun, Lin Tianhai, Liu Fang, Li Guisen, Yang Hao, Zhang Yong
Institutes for Systems Genetics, National Health Commission (NHC) Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China.
Department of Thoracic Surgery, Institute of Thoracic Oncology, West China Hospital, Sichuan University, Chengdu, China.
Front Chem. 2022 Feb 24;10:839470. doi: 10.3389/fchem.2022.839470. eCollection 2022.
Site-specific N-glycosylation characterization requires intact N-glycopeptide analysis based on suitable tandem mass spectrometry (MS/MS) method. Electron-transfer/higher-energy collisional dissociation (EThcD), stepped collision energy/higher-energy collisional dissociation (sceHCD), higher-energy collisional dissociation-product-dependent electron-transfer dissociation (HCD-pd-ETD), and a hybrid mass spectrometry fragmentation method EThcD-sceHCD have emerged as valuable approaches for glycoprotein analysis. However, each of them incurs some compromise, necessitating the systematic performance comparisons when applied to the analysis of complex clinical samples (e.g., plasma, urine, cells, and tissues). Herein, we compared the performance of EThcD-sceHCD with those previous approaches (EThcD, sceHCD, HCD-pd-ETD, and sceHCD-pd-ETD) in the intact N-glycopeptide analysis, and determined its applicability for clinical N-glycoproteomic study. The intact N-glycopeptides of distinct samples, namely, plasma from prostate cancer (PCa) patients, urine from immunoglobulin A nephropathy (IgAN) patients, human hepatocarcinoma cell line (HepG2), and thyroid tissues from thyroid cancer (TC) patients were analyzed by these methods. We found that EThcD-sceHCD outperformed other methods in the balance of depth and accuracy of intact N-glycopeptide identification, and sceHCD and EThcD-sceHCD have good complementarity. EThcD-sceHCD holds great potential for biomarker discovery from clinical samples.
位点特异性N-糖基化表征需要基于合适的串联质谱（MS/MS）方法进行完整的N-糖肽分析。电子转移/高能碰撞解离（EThcD）、阶梯碰撞能量/高能碰撞解离（sceHCD）、高能碰撞解离-产物依赖性电子转移解离（HCD-pd-ETD）以及一种混合质谱碎裂方法EThcD-sceHCD已成为糖蛋白分析的有价值方法。然而，它们各自都有一些折衷之处，在应用于复杂临床样本（如血浆、尿液、细胞和组织）分析时需要进行系统的性能比较。在此，我们在完整N-糖肽分析中比较了EThcD-sceHCD与那些先前方法（EThcD、sceHCD、HCD-pd-ETD和sceHCD-pd-ETD）的性能，并确定了其在临床N-糖蛋白质组学研究中的适用性。通过这些方法分析了不同样本的完整N-糖肽，即前列腺癌（PCa）患者的血浆、免疫球蛋白A肾病（IgAN）患者的尿液、人肝癌细胞系（HepG2）以及甲状腺癌（TC）患者的甲状腺组织。我们发现，EThcD-sceHCD在完整N-糖肽鉴定的深度和准确性平衡方面优于其他方法，并且sceHCD和EThcD-sceHCD具有良好的互补性。EThcD-sceHCD在从临床样本中发现生物标志物方面具有巨大潜力。
