Quantitative Analysis of α-1-Antitrypsin Glycosylation Isoforms in HCC Patients Using LC-HCD-PRM-MS
Quantitative Analysis of α-1-Antitrypsin Glycosylation Isoforms in HCC Patients Using LC-HCD-PRM-MS
复制标题
使用 LC-HCD-PRM-MS 定量分析 HCC 患者中的 Alpha-1-抗胰蛋白酶糖基化亚型
DOI：
10.1021/acs.analchem.0c00420
复制
发表时间：
2020-06-16
影响因子：
7.4
通讯作者：
Lubman, David M.
中科院分区：
文献类型：
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作者：
Yin, Haidi;Zhu, Jianhui;Lubman, David M.
血清蛋白糖基化的变化通常与各种疾病的发展有关，因此可用于诊断。本研究采用液相色谱-串联质谱法对早期HCC和肝硬化患者血清α -1-抗胰蛋白酶（A1AT）的位点特异性糖型进行精确的结构分析和定量。采用抗A1AT抗体偶联琼脂糖珠免疫沉淀法从患者血清中纯化血清蛋白A1AT，分离得到的A1AT蛋白经LC-MS/MS酶切分析。本研究整合了两种串联质谱分析策略：用于A1AT糖肽结构分析的非靶向阶梯HCD策略和用于定量HCC和肝硬化患者血清中A1AT位点特异性糖型的靶向平行反应监测（PRM）策略。因此，采用pGlyco2.0软件进行糖肽鉴定，采用Skyline软件在MS/MS光谱中用Y1离子（肽+GlcNAc）进行糖型定量。采用HCD-MS/MS对患者样本中的10个A1AT位点特异性糖肽进行鉴定，其中7个在患者样本中采用HCD-PRM-MS进一步定量。我们发现我们的策略能够区分糖肽的异构体，其中几种异构体在肝硬化和HCC患者之间表现出明显不同的模式。我们还发现A1AT的一个糖肽不同电荷态的比值（2+/3+）可以显著区分早期HCC和肝硬化，其受试者工作特征曲线下面积AUC为0.9。进一步分析表明，这种差异可能与多糖基序的唾液酸/半乳糖连锁有关。
The change in glycosylation of serum proteins is often associated with the development of various diseases and thus can be used for diagnosis. In this study, a liquid chromatography-tandem mass spectrometry-based method is used for accurate structural analysis and quantification of site-specific glycoforms of serum alpha-1-antitrypsin (A1AT) in early-stage HCC and cirrhosis patients. Serum protein A1AT was purified from patient sera by immunoprecipitation with anti-A1AT antibody conjugated agarose beads, and the isolated A1AT protein was digested and analyzed by LC-MS/MS. Two tandem mass spectrometry strategies are integrated in this study: a nontargeted stepped HCD strategy for structural analysis of A1AT glycopeptides and a targeted parallel reaction monitoring (PRM) strategy for quantification of site-specific glycoforms of A1AT in HCC and cirrhosis patient sera. Accordingly, pGlyco2.0 software was used for glycopeptide identification, and Skyline software was used for glycoform quantification using the Y1 ion (peptide+GlcNAc) in MS/MS spectra. Ten site-specific glycopeptides of A1AT were identified with stepped HCD-MS/MS in patient samples, 7 of which were further quantified using HCD-PRM-MS among patient samples. We found that our strategy was able to distinguish isomers of glycopeptides where several isomers showed distinctly different patterns between cirrhosis and HCC patients. We also found that the ratio of different charge states (2+/3+) of one glycopeptide of A1AT can significantly discriminate early-stage HCC from cirrhosis with the area under the receiver operating characteristic curve AUC of 0.9. Further analysis showed that the difference may be related to the sialic acid/galactose linkage of the glycan motif.