基于Fe<sub>3</sub>O<sub>4</sub>/乙二胺四乙酸磁性粒子的集成化蛋白质组学方法

引用本文: 唐君, 郭凯珠, 陈文东, 宋培培, 封顺, 胡巢凤, 许瑞莲, 田瑞军. 基于Fe₃O₄/乙二胺四乙酸磁性粒子的集成化蛋白质组学方法[J]. 色谱, 2016, 34(12): 1264-1270. doi: 10.3724/SP.J.1123.2016.09033 shu

Citation: TANG Jun, GUO Kaizhu, CHENG Wendong, SONG Peipei, FENG Shun, HU Chaofeng, XU Ruilian, TIAN Ruijun. Fe₃O₄/ethylenediaminetetraacetic acid magnetic beads-based integrated method for proteomic analysis[J]. Chinese Journal of Chromatography, 2016, 34(12): 1264-1270. doi: 10.3724/SP.J.1123.2016.09033 shu

基于Fe₃O₄/乙二胺四乙酸磁性粒子的集成化蛋白质组学方法

1.
深圳市人民医院肿瘤研究所, 广东深圳 518020;
2.
南方科技大学化学系, 广东深圳 518055;
3.
西南交通大学生命科学与工程学院, 四川成都 610031;
4.
深圳市细胞微环境重点实验室, 广东深圳 518055;
5.
暨南大学基础医学院病理生理学系, 广东广州 510632
基金项目:
国家自然科学基金项目（21575057）；深圳市科技计划项目（JCYJ20150901153557178，JSGG20160301103415523）.

摘要: 建立了基于Fe₃O₄/乙二胺四乙酸（EDTA）磁性粒子的集成化蛋白质组学研究方法。首先用共沉淀法合成EDTA负载的Fe₃O₄/EDTA磁性粒子。在优化的溶液条件下（95%乙腈-1%三氟乙酸，体积分数），100 μ g Fe₃O₄/EDTA磁性粒子可吸附12.4 μ g牛血清白蛋白（BSA），吸附容量是商品化磁珠的10倍左右。以BSA作为标准蛋白质，对所合成的Fe₃O₄/EDTA磁性粒子作为蛋白质组学反应器的酶解时间进行了优化，发现Fe₃O₄/EDTA磁性粒子处理BSA酶解1、8和16 h的肽段序列覆盖率和特征肽段结果相当。因此，可以将复杂的蛋白质样品前处理时间缩短至2 h内。最后，将所合成的Fe₃O₄/EDTA磁性粒子应用于血清的蛋白质组学研究，成功地鉴定出218种蛋白质，其中包含了41种美国食品药品管理局（FDA）认证的生物标志物。所发展的基于Fe₃O₄/EDTA磁性粒子的蛋白质组学样品前处理方法将蛋白质样品预富集、还原、烷基化、酶解、多肽除盐和洗脱等步骤集成到一起，减少了样品转移和处理所造成的损失。这种技术具有快速、灵敏和易于操作的特点，可用于临床蛋白质组学研究。

关键词:

English

Fe₃O₄/ethylenediaminetetraacetic acid magnetic beads-based integrated method for proteomic analysis

1.
Institute of Oncology, Shenzhen People's Hospital, Shenzhen 518020, China;
2.
Department of Chemistry, South University of Science and Technology of China, Shenzhen 518055, China;
3.
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China;
4.
Shenzhen Key Laboratory of Cell Microenvironment, Shenzhen 518055, China;
5.
Department of Pathophysiology, School of Basic Medicine, Jinan University, Guangzhou 510632, China
Received Date: 15 September 2016
Fund Project:
National Natural Science Foundation of China (No. 21575057)

Abstract: A Fe₃O₄/EDTA (ethylenediaminetetraacetic acid) magnetic beads-based integrated method for proteomic analysis was developed. Fe₃O₄ magnetic beads loaded with EDTA were prepared firstly via co-precipitation method. One hundred μ g Fe₃O₄/EDTA magnetic beads could adsorb 12.4 μ g BSA (bovine serum albumin) in the optimized solution (95% acetonitrile-1% trifluoracetic acid, v/v). The binding capacity was about 10 times higher than that of the commercialized magnetic beads. The digestion time of Fe₃O₄/EDTA magnetic beads as the proteomic reactor was also optimized using BSA as the standard protein. The results indicated that the peptide coverage and unique peptides obtained from Fe₃O₄/EDTA magnetic beads with digestion time of 1, 8 and 16 h were comparable. We then applied the Fe₃O₄/EDTA magnetic beads for the serum proteome analysis. A total of 218 proteins were identified successfully, including 41 biomarkers approved by Food and Drug Administration (FDA). Protein preconcentration, reduction, alkylation, digestion, peptide desalting, and elution can be achieved in an integrated manner by the Fe₃O₄/EDTA magnetic beads-based proteomics sample preparation, which reduced the sample loss during sample transfering and processing. The developed method is fast, sensitive and easy to operate, which has prospective application for the clinic proteomics research.

Key words:

Fe₃O₄/EDTA (ethylenediaminetetraacetic acid)
/ magnetic beads
/ proteomics
/ sample preparation

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基于Fe₃O₄/乙二胺四乙酸磁性粒子的集成化蛋白质组学方法

English

Fe₃O₄/ethylenediaminetetraacetic acid magnetic beads-based integrated method for proteomic analysis

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基于Fe3O4/乙二胺四乙酸磁性粒子的集成化蛋白质组学方法

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Fe3O4/ethylenediaminetetraacetic acid magnetic beads-based integrated method for proteomic analysis

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通讯作者: 陈斌, bchen63@163.com

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Fe₃O₄/ethylenediaminetetraacetic acid magnetic beads-based integrated method for proteomic analysis

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