The Hong Kong Polytechnic University Shenzhen Research Institute, Shenzhen, People's Republic of China.
Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong.
Anal Chem. 2020 Jun 16;92(12):8201-8208. doi: 10.1021/acs.analchem.0c00420. Epub 2020 Jun 2.
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 α-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.
血清蛋白糖基化的改变通常与各种疾病的发展有关，因此可用于诊断。在本研究中，采用基于液相色谱-串联质谱的方法，对早期 HCC 和肝硬化患者血清α-1-抗胰蛋白酶（A1AT）的特异性糖型进行准确的结构分析和定量。通过用抗 A1AT 抗体偶联琼脂糖珠对患者血清中的血清蛋白 A1AT 进行免疫沉淀，从患者血清中纯化血清 A1AT 蛋白，然后用 LC-MS/MS 对分离的 A1AT 蛋白进行消化和分析。本研究整合了两种串联质谱策略：一种非靶向分步 HCD 策略用于 A1AT 糖肽的结构分析，另一种靶向平行反应监测（PRM）策略用于定量 HCC 和肝硬化患者血清中 A1AT 的特异性糖型。相应地，使用 pGlyco2.0 软件进行糖肽鉴定，使用 Skyline 软件在 MS/MS 谱中使用 Y1 离子（肽+GlcNAc）进行糖型定量。在患者样本中，通过分步 HCD-MS/MS 鉴定了 A1AT 的 10 个特异性糖肽，其中 7 个在患者样本中进一步通过 HCD-PRM-MS 进行定量。我们发现我们的策略能够区分糖肽的异构体，其中一些异构体在肝硬化和 HCC 患者之间表现出明显不同的模式。我们还发现，A1AT 的一种糖肽的不同电荷状态（2+/3+）的比例可以显著区分早期 HCC 与肝硬化，ROC 曲线下面积 AUC 为 0.9。进一步分析表明，这种差异可能与聚糖基序的唾液酸/半乳糖连接有关。
