色谱 ›› 2017, Vol. 35 ›› Issue (3): 280-285.DOI: 10.3724/SP.J.1123.2016.10012

• 研究论文 • 上一篇    下一篇

应用随机寡核苷酸文库修饰磁性颗粒提高血浆蛋白质的鉴定覆盖度

邓楠1, 梁振2, 边阳阳3, 张明建1, 张柯1, 张丽华2, 张玉奎2   

  1. 1. 中国烟草总公司郑州烟草研究院, 河南 郑州 450001;
    2. 中国科学院大连化学物理研究所, 中科院分离分析化学重点实验室, 国家色谱研究分析中心, 辽宁 大连 116023;
    3. 郑州大学第一附属医院医学研究中心, 河南 郑州 450052
  • 收稿日期:2016-10-09 出版日期:2017-03-08 发布日期:2013-07-16
  • 通讯作者: 张丽华,Tel:(0411)84379720,E-mail:lihuazhang@dicp.ac.cn.
  • 基金资助:

    国家自然科学基金项目(21505158,81600046).

Improved human plasma identification coverage based on random oligonucleotide library immobilized magnetic particles

DENG Nan1, LIANG Zhen2, BIAN Yangyang3, ZHANG Mingjian1, ZHANG Ke1, ZHANG Lihua2, ZHANG Yukui2   

  1. 1. Zhengzhou Tobacco Research Institute of China National Tobacco Corporation, Zhengzhou 450001, China;
    2. Dalian Institute of Chemical Physics, Key laboratory of Separation Sciences for Analytical Chemistry, Chinese Academy of Sciences; National Chromatographic Research and Analysis Center, Dalian 116023, China;
    3. Medical Research Centre, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
  • Received:2016-10-09 Online:2017-03-08 Published:2013-07-16
  • Supported by:

    National Natural Science Foundation of China (Nos. 21505158, 81600046).

摘要:

基于随机寡核苷酸与蛋白质之间的离子、亲和、疏水、氢键等相互作用力及多种空间结构作用,发展了一种基于随机寡核苷酸文库作为配基的新型血浆样品处理方法。采用寡核苷酸文库修饰的磁性颗粒(MNP@ssDNA)材料,在生理缓冲体系条件下捕获血浆样品中的蛋白质。比较了两种洗脱体系的洗脱效果,并利用nano-RPLC-ESI-MS/MS对获得的蛋白质酶解液组分进行分析。结果表明,MNP@ssDNA材料处理后的血浆蛋白质鉴定数量提升了约29.5%,两种洗脱体系呈现良好的互补性(26.7%)。血浆中前10种高丰度蛋白质的谱图占有率从处理前的31.82%降低到21.31%(洗脱体系1)和26.20%(洗脱体系2)。在鉴定到的蛋白质中,丰度最低的蛋白质在血浆中的质量浓度约为0.29 ng/mL,该蛋白质仅在MNP@ssDNA材料处理后被鉴定到。结果证明MNP@ssDNA策略不仅能有效降低血浆中高丰度蛋白质的丰度,也为低丰度蛋白质的深度挖掘提供了新的思路。

关键词: 蛋白质, 低丰度, 高丰度, 谱图数量, 随机寡核苷酸文库, 血浆

Abstract:

A novel human plasma proteome sample pretreatment strategy was developed based on the interaction of random oligonucleotides with human plasma proteins, such as ionic interaction, affinity interaction, hydrophobic interaction, hydrogen bonding or spatial structure and so on. Random oligonucleotide library was immobilized on the magnetic particles by biotin-avidin interaction, and dispersed in 20 mmol/L Tris-HCl buffer (pH 7.4), followed by incubation with plasma proteins. Two elution systems were used to elute the proteins interacted with random oligonucleotides, separately. Nano-RPLC-ESI-MS/MS analysis was performed for protein identification. The number of proteins identified after treatment was increased by 29.5%, and two elution systems displayed good complementarity (26.7%). The total ratio of spectral counts of the top ten high abundant protein in human plasma was decreased from 31.82% to 21.31% (elution system 1) and 26.20% (elution system 2). In all the proteins identified, the lowest abundant protein (0.29 ng/mL) was only identified after magnetic nanoparticles@single-stranded DNA (MNP@ssDNA) treatment, which demonstrated that this strategy not only decreased the abundance of highly abundant proteins, but also provided a new idea for digging more lowly abundant proteins.

Key words: high abundance, low abundance, plasma, protein, random oligonucleotide library, spectral count

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