Acta Chimica Sinica ›› 2013, Vol. 71 ›› Issue (03): 343-346.DOI: 10.6023/A12121103 Previous Articles     Next Articles

Communications

氧化铝富集糖肽的研究

赵旭a, 姜武辉c, 于龙b, 邹丽娟a, 李秀玲b, 梁鑫淼b   

  1. a 大连医科大学附属第二医院 大连 116023;
    b 中国科学院大连化学物理研究所 中国科学院分离分析化学重点实验室 大连 116023;
    c 辽宁宇洁环保咨询有限公司 沈阳 110000
  • 收稿日期:2012-12-26 出版日期:2013-03-14 发布日期:2013-01-25
  • 通讯作者: 邹丽娟,李秀玲 E-mail:zoulijuan1963@sina.com;lixiuling@dicp.ac.cn
  • 基金资助:

    项目受国家高技术研究发展计划863计划(No.2012AA020203)、国家自然科学基金(Nos.81171486,21105100)和材料复合新技术国家重点实验室(武汉理工大学,2013-KF-8)开放基金资助.

Selective Enrichment of Glycopeptides Using Aluminum Oxide

Zhao Xua, Jiang Wuhuic, Yu Longb, Zou Lijuana, Li Xiulingb, Liang Xinmiaob   

  1. a The Second Hospital Affiliated to Dalian Medical University, Dalian 116023;
    b Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023;
    c Liaoning Yu Jie Environmental Consulting Limited Company, Shenyang 110000
  • Received:2012-12-26 Online:2013-03-14 Published:2013-01-25
  • Supported by:

    Project supported by the National High Technology Research and Development Program 863 (No. 2012AA020203), the National Natural Science Foundation of China (Nos. 81171486, 21105100) and State Key Laboratory of Advanced Materials Synthesis Processing (Wuhan University of Technology, 2013-KF-8).

Protein glycosylation is significantly associated with cells cycle, immune regulation, cells recognition and cells adhesion. Aberrant glycosylation expression is involved in many diseases, such as rheumatoid arthritis, cancer, and chronic obstructive pulmonary disease. Thus, characterization of the protein glycosylation is very important to understand the life process. However, detection of glycopeptides is difficult by mass spectrometry because of the low concentration in complex sample and the suppressed signal by non-glycopeptides. Therefore, it is essential to explore an effective technique for glycopeptide enrichment. In this study, an alumina based-materials was used to selectively enrich glycopeptides. Firstly, we investigated the retention mechanism by changing the concentration of acetonitrile (ACN). With the decreased concentration of ACN, it was found that peptides were eluted according to their hydrophilicity. Moreover, most of the non-glycopeptides were eluted earlier than the glycopeptides in the high concentration of ACN fraction and the glycopeptides were found in the low concentration of ACN fraction. This result proved that hydrophilic interaction is one of the retention mechanisms for peptides retained by alumina. Secondly, we investigated the retention mechanism by changing the concentration of ammonium hydroxide. ACN were fixed at a high concentration and peptides were subsequently eluted with different concentration of ammonium hydroxide solution. At a low concentration of ammonium hydroxide solution, the peptides were no found. But at a high concentration of ammonium hydroxide solution, the glycopeptides and non-glycopeptides were eluted simultaneously. This proved that the ligand exchange is also one of the retention mechanisms, where the retention of the glycopeptides and non-glycopeptides showed no different under this mechanism. Based on the above mentioned results, the enrichment condition was optimized under the model of solid-phase extraction. High concentration of ACN mixed with a certain concentration of ammonium hydroxide solution as loading buffer and low concentration of ACN mixed with a certain concentration of ammonium hydroxide solution as elution buffer. The number of glycopeptides enriched by alumina were compared with glycopeptides before enrichment and enriched by Sepharose. In the tryptic HRP digest, 7 glycopeptides were found before enrichment, 16 glycopeptides were found by alumina and 14 glycopeptides were found by Sepharose. In the tryptic IgG digest, 2 glycopeptides were found before enrichment, 12 glycopeptides were found by alumina and 4 glycopeptides were found by Sepharose. In conclusion, alumina based-method used to enrich glycopeptides has high selection and wide coverage.

Key words: alumina, glycopeptides, enrichment, mass spectrometry, peptides