高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (6): 1141-1149.doi: 10.7503/cjcu20180782

• 分析化学 • 上一篇    下一篇

质谱法定量测定高密度脂蛋白结合蛋白的糖基化水平

魏王慧1, 储艳秋2(), 陈鹰1, 高艳秋1, 丁传凡2()   

  1. 1. 上海计量测试技术研究院, 上海 201203
    2. 复旦大学化学系, 上海 200438
  • 收稿日期:2018-11-21 出版日期:2019-06-10 发布日期:2019-04-04
  • 作者简介:

    联系人简介: 储艳秋, 男, 博士, 副教授, 主要从事有机分子和生物大分子非共价作用的质谱方面的研究. E-mail: chuyq@fudan.edu.cn;丁传凡, 男, 博士, 教授, 博士生导师, 主要从事质谱方面的研究. E-mail: cfding@fudan.edu.cn

  • 基金资助:
    国家自然科学基金(批准号: 21773035)资助.

Quantitative Determination of the Glycosylation Level for the Binding Proteins to High Density Lipoprotein by Mass Spectrometry

WEI Wanghui1, CHU Yanqiu2(), CHEN Ying1, Gao Yanqiu1, DING Chuanfan2()   

  1. 1. Shanghai Institute of Measurement and Testing Technology, Shanghai 201203, China
    2. Chemistry Department, Fudan University, Shanghai 200438, China
  • Received:2018-11-21 Online:2019-06-10 Published:2019-04-04
  • Supported by:
    † Supported by the National Natural Science of Foundation of China(No.21773035).

摘要:

采用质谱法对4种高密度脂蛋白(HDL)的结合蛋白重组人载脂蛋白血清淀粉样蛋白A(SAA)、 α1-抗胰蛋白酶(A1AT)、 α2-人体血清糖蛋白(A2HSG)和A载脂蛋白C3(Apo C3)从蛋白质含量(蛋白的绝对定量)、 位点特异性糖基化(糖肽的相对定量)及聚糖位点占有率等方面进行了研究. 利用四极杆-飞行时间质谱仪(Q-TOF)测量糖蛋白标样酶解产物的二级质谱碎片离子, 用Byonic软件发现了新的糖基化位点信息, 即增加了原位点处聚糖糖型的种类. 对于A2HSG, 新增了N-糖基化156位点上的4种糖型, N-糖基化176位点上的6种糖型, O-糖基化319位点的4种O-聚糖和O-糖基化346位点上的1种糖型. 对于Apo C3, 只有O-糖基化94一个位点, 在此位点上新增了9种糖型. 同时, 调整了用于定量蛋白的多肽, 使得定量更加准确. 采用三重四极杆串级质谱仪(UPLC-ESI-QQQ)研究了4种结合蛋白中多肽和糖肽的多反应监测(MRM)行为, 并重新计算了每种聚糖的位点占有率, 优化了现有的定量方法.

关键词: 质谱法, 高密度脂蛋白的结合蛋白, 定点糖基化, 新糖型, 多反应监测

Abstract:

The protein content, site-specific glycosylation, and site occupancy of glycan and so on for serum amyloid A(SAA), α-1-antitrypsin(A1AT), α-2-HS-glycoprotein(A2HSG) and apolipoprotein C-Ⅲ(Apo C3) bound to high density lipoproteins(HDL) were investigated by mass spectrometry. Four kinds of trypsin digested protein standard samples were fragmented in liquid phase-quadrupole time-of-flight mass spectrometer. Some new glycosylation sites and new glycoforms were found by analyzing and matching fragment ions. For A2HSG, one O-glycosylation site and four glycoforms at that site were found, as well as 11 glycoforms at the original three sites. For Apo C3, 9 glycoforms at the original site were founded. Meanwhile, a unique peptide that used to quantify the protein were adjusted to make the quantification more accurate. Moreover, the multiple reaction monitor(MRM) behavior of peptides and glycopeptides in four proteins were studied and the site occupancy rate of each glycoform were recalculated. It is expected that our new findings in the process of exploration of site-specific glycosylation of HDL bound SAA, A1AT, A2HSG and Apo C3 will provide valuable informations in search of new HDL biomarks and will probably be practically applied in clinical detection of diseases related to HDL.

Key words: Mass spectrometry, Binding proteins to high density lipoprotein, Site-specific glycosylation, New glycoform, Multiple reaction monitoring

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