Lin Yu, Zhang Jie, Arroyo Ana, Singal Amit G, Parikh Neehar D, Lubman David M
Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, United States.
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States.
Front Oncol. 2022 Mar 17;12:818001. doi: 10.3389/fonc.2022.818001. eCollection 2022.
Aberrant specific N-glycosylation, especially the increase in fucosylation on specific peptide sites of serum proteins have been investigated as potential markers for diagnosis of nonalcoholic steatohepatitis (NASH)-related HCC. We have combined a workflow involving broad scale marker discovery in serum followed by targeted marker evaluation of these fucosylated glycopeptides. This workflow involved an LC-Stepped HCD-DDA-MS/MS method coupled with offline peptide fractionation for large-scale identification of -glycopeptides directly from pooled serum samples (each n=10) as well as differential determination of N-glycosylation changes between disease states. We then evaluated the fucosylation level of the glycoprotein ceruloplasmin among 62 patient samples (35 cirrhosis, 27 early-stage NASH HCC) by LC-Stepped HCD-PRM-MS/MS to quantitatively analyze 18 targeted glycopeptides. Of these targets, we found the ratio of fucosylation of a tri-antennary glycopeptide from site N762, involving N762_ HexNAc(5)Hex(6)Fuc(2)NeuAc(3) (P=0.0486), increased significantly from cirrhosis to early HCC. This fucosylation ratio of a tri-antennary glycopeptide in CERU could be a potential biomarker for further validation in a larger sample set and could be a promising candidate for early detection of NASH HCC.
异常的特异性N-糖基化，尤其是血清蛋白特定肽位点上岩藻糖基化的增加，已被研究作为非酒精性脂肪性肝炎（NASH）相关肝癌诊断的潜在标志物。我们结合了一个工作流程，包括在血清中进行大规模标志物发现，然后对这些岩藻糖基化糖肽进行靶向标志物评估。该工作流程涉及一种LC-阶梯式HCD-DDA-MS/MS方法，结合离线肽分级分离，用于直接从合并的血清样本（每组n = 10）中大规模鉴定N-糖肽，以及疾病状态之间N-糖基化变化的差异测定。然后，我们通过LC-阶梯式HCD-PRM-MS/MS评估了62例患者样本（35例肝硬化，27例早期NASH肝癌）中铜蓝蛋白的岩藻糖基化水平，以定量分析18个靶向糖肽。在这些靶点中，我们发现来自位点N762的三触角糖肽的岩藻糖基化比率，即N762_HexNAc(5)Hex(6)Fuc(2)NeuAc(3)（P = 0.0486），从肝硬化到早期肝癌显著增加。CERU中三触角糖肽的这种岩藻糖基化比率可能是一种潜在的生物标志物，有待在更大的样本集中进一步验证，并且可能是早期检测NASH肝癌的有希望的候选物。
