A Panel of Glycopeptides as Candidate Biomarkers for Early Diagnosis of NASH Hepatocellular Carcinoma Using a Stepped HCD Method and PRM Evaluation.
A Panel of Glycopeptides as Candidate Biomarkers for Early Diagnosis of NASH Hepatocellular Carcinoma Using a Stepped HCD Method and PRM Evaluation.
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DOI：
10.1021/acs.jproteome.1c00175
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发表时间：
2021-06-04
影响因子：
4.4
通讯作者：
Lubman DM
中科院分区：
文献类型：
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作者：
Lin Y;Zhu J;Pan L;Zhang J;Tan Z;Olivares J;Singal AG;Parikh ND;Lubman DM
血清蛋白特定肽位点的n -糖基化变化已被研究作为诊断非酒精性脂肪性肝炎（NASH）相关HCC的潜在标志物。为了完成这项工作，我们结合了一种新的工作流程，包括在血清中大规模发现标记物，然后对这些糖肽进行靶向标记物评估。工作流程包括lc - step HCD-DDA-MS/MS方法与离线肽分离相结合，用于直接从合并的血清样本（每个n = 10）中大规模鉴定n -糖基化，以及差异测定n -糖基化在疾病状态之间的变化。然后，我们通过lc - step HCD-PRM-MS/MS对78例个体患者样本（40例肝硬化，28例早期NASH HCC和10例晚期NASH HCC）中的几种潜在诊断n -糖肽进行了评估，定量分析了来自7种糖蛋白的65种靶向糖肽。在这些靶点中，我们发现来自VTNC的位点特异性n -糖肽n169GSLFAFR_HexNAc(4)Hex(5)NeuAc(2)和n242ISDGFDGIPDNVDAALALPAHSYSGR_HexNAc(5)Hex(6)Fuc(1)NeuAc(3)与NASH肝硬化和NASH HCC患者的样本相比显著增加（p < 0.05）。将这两种糖肽的检测结果与AFP联合使用时，ROC曲线分析显示，与单独使用AFP相比，AUC值分别增加至0.834 （95% CI, 0.748 ~ 0.921）和0.847 (95% CI, 0.766 ~ 0.932) （AUC = 0.791, 95% CI, 0.690 ~ 0.892）。这两种糖肽可能作为NASH相关肝硬化患者早期HCC诊断的潜在生物标志物。
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.