A Panel of Glycopeptides as Candidate Biomarkers for Early Diagnosis of NASH Hepatocellular Carcinoma Using a Stepped HCD Method and PRM Evaluation

Changes
in N-glycosylation on specific peptide sites of serum proteins
have been investigated as potential markers for diagnosis of nonalcoholic
steatohepatitis (NASH)-related HCC. To accomplish this work, a novel
workflow involving broad-scale marker discovery in serum followed
by targeted marker evaluation of these glycopeptides were combined.
The workflow involved an LC-Stepped HCD-DDA-MS/MS method coupled with
offline peptide fractionation for large-scale identification of N-glycopeptides directly from pooled serum samples (each n = 10) as well as differential determination of N-glycosylation
changes between disease states. We then evaluated several potentially
diagnostic N-glycopeptides among 78 individual patient samples (40
cirrhosis, 28 early stage NASH HCC, and 10 late-stage NASH HCC) by
LC-Stepped HCD-PRM-MS/MS to quantitatively analyze 65 targeted glycopeptides
from 7 glycoproteins. Of these targets, we found site-specific N-glycopeptides n169GSLFAFR_HexNAc­(4)­Hex­(5)­NeuAc­(2)
and n242ISDGFD­GIPDNV­DAALAL­PAHSY­SGR_Hex­NAc­(5)­Hex­(6)­Fuc­(1)­NeuAc­(3)
from VTNC were significantly increased comparing samples from patients
with NASH cirrhosis and NASH HCC (p < 0.05). When
combining results of these 2 glycopeptides with AFP, the ROC curve
analysis demonstrated the AUC value increased to 0.834 (95% CI, 0.748–0.921)
and 0.847 (95% CI, 0.766–0.932), respectively, as compared
to that of AFP alone (AUC = 0.791, 95% CI, 0.690–0.892). These
2 glycopeptides may serve as potential biomarkers for early HCC diagnosis
in patients with NASH related cirrhosis.

针对非酒精性脂肪性肝炎（NASH）相关肝细胞癌（HCC）的诊断，研究者们对血清蛋白特定肽位点的N-糖基化变化进行了探究，将其视为潜在的生物标志物。为实现此研究目标，结合了一种创新的工作流程，该流程首先在血清中进行大规模标志物发现，随后对这些糖肽进行靶向标志物评估。该工作流程采用液相色谱（LC）逐步高能碰撞解离-电喷雾电离-串联质谱（HCD-DDA-MS/MS）方法，并辅以离线肽段分级分离技术，以实现对混合血清样本（每个样本n=10）中N-糖肽的大规模鉴定，以及对疾病状态下N-糖基化变化的差异分析。随后，通过LC逐步高能碰撞解离-正离子反应模式-串联质谱（HCD-PRM-MS/MS）技术，对来自7种糖蛋白的65个靶向糖肽进行了定量分析。在这些目标中，我们发现来自VTNC的n169GSLFAFR_HexNAc(4)Hex(5)NeuAc(2)和n242ISDGFDGIPDNVDAALALPAHSYSGR_HexNAc(5)Hex(6)Fuc(1)NeuAc(3)两个位点特异性N-糖肽在NASH肝硬化与NASH HCC患者样本中显著增加（p<0.05）。当将这两个糖肽的结果与甲胎蛋白（AFP）结合时，ROC曲线分析显示AUC值分别增加至0.834（95% CI，0.748–0.921）和0.847（95% CI，0.766–0.932），与单独使用AFP相比（AUC=0.791，95% CI，0.690–0.892），显示出显著提升。这两类糖肽有望作为NASH相关肝硬化患者早期HCC诊断的潜在生物标志物。