离子交换色谱-基质辅助激光解吸电离串联飞行时间质谱分离鉴定中国人乳中的β-酪蛋白

doi:10.3724/SP.J.1123.2013.01010

色谱 ›› 2013, Vol. 31 ›› Issue (5): 429-434.DOI: 10.3724/SP.J.1123.2013.01010

离子交换色谱-基质辅助激光解吸电离串联飞行时间质谱分离鉴定中国人乳中的β-酪蛋白

黄宇^1,2, 任皓威^1,2*, 刘彪³, 刘宁^1,2*, 李萌^1,2, 王东茂⁴

1. 国家乳业工程技术研究中心, 黑龙江省乳品工业技术开发中心, 黑龙江哈尔滨 150028;
2. 东北农业大学食品学院, 乳品科学教育部重点实验室, 黑龙江哈尔滨 150030;
3. 内蒙古伊利实业集团股份有限公司, 内蒙古呼和浩特 010110;
4. 北京蛋白质组研究中心, 北京 102206

收稿日期:2012-12-31 修回日期:2013-03-05 出版日期:2013-05-28 发布日期:2013-05-16
通讯作者: 任皓威,E-mail:renhw800903@yahoo.com.cn;刘宁,E-mail:ningliu6666@yahoo.com.cn.
基金资助:
"十二五"国家科技支撑计划项目(2011BAD09B03).

Separation and identification of β-casein from Chinese human milk by ion exchange chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry

HUANG Yu^1,2, REN Haowei^1,2*, LIU Biao³, LIU Ning^1,2*, LI Meng^1,2, WANG Dongmao⁴

1. National Dairy Engineering & Technical Research Center, Heilongjiang Dairy Industry Technical Development Center, Harbin 150028, China;
2. College of Food Science, Northeast Agricultural University, Key Laboratory of Dairy Science, Ministry of Education, Harbin 150030, China;
3. Inner Mongolia Yili Industrial Group Co., Ltd., Hohhot 010110, China;
4. Beijing Proteome Research Center, Beijing 102206, China

Received:2012-12-31 Revised:2013-03-05 Online:2013-05-28 Published:2013-05-16

摘要/Abstract

摘要：

以新鲜中国人乳为研究对象,在酸性条件下加氯化钙分离酪蛋白,用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法测定分离效果。从pH值、离心力和CaCl₂浓度等3个方面对分离效果进行评价。结果表明:当pH=4.3,离心力为10400 g, CaCl₂溶液浓度为60 mmol/L时,分离得到的酪蛋白组分纯度最高。对所得酪蛋白沉淀进行DEAE阴离子交换色谱分离,得到3个洗脱液。对其中第3个峰进行Western-Blot法及基质辅助激光解吸电离串联飞行时间质谱法(MALDI-TOF/TOF MS)鉴定,证明该峰能与人β-酪蛋白抗体特异性结合,确认其为人乳β-酪蛋白;将质谱结果在Mascot数据库中进行数据检索,序列覆盖率为50%,进一步确定其为人乳β-酪蛋白。综上所述,我们建立了能够得到符合蛋白质组学研究要求的人乳β-酪蛋白的方法。

关键词: β-酪蛋白, 纯化, 阴离子交换色谱, 质谱, 中国人乳

Abstract:

The selective precipitation of whole casein from skimmed milk was achieved by the addition of calcium salt under acidic pH. The effects of pH, centrifugal force and final concentration of CaCl₂ on the separation of casein were studied by measuring the purity of final products using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that casein with the highest purity could be obtained with the pH of 4.3, the centrifugal force of 10 400 g and the final concentration of CaCl₂ of 60 mmol/L. The casein was processed with DEAE anion exchange chromatography and three peaks were obtained. Then the third peak (peak Ⅲ) was identified with Western-Blot method and matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). The identification of Western-Blot showed that peak Ⅲ can combine with the specificity of human milk β-casein antibody, and it is proved to be human milk β-casein. The fingerprints of peak Ⅲ were analyzed by Mascot searching, and the sequence coverage was 50%, further supporting it is human milk β-casein. In conclusion, an effective method to obtain human milk β-casein from milk samples through DEAE anion exchange chromatography was established, and it is suitable for the proteomics research requirements of the β-casein from human milk.

Key words: β-casein, anion exchange chromatography, Chinese human milk, mass spectrometry, purification

中图分类号:

O658

黄宇, 任皓威, 刘彪, 刘宁, 李萌, 王东茂. 离子交换色谱-基质辅助激光解吸电离串联飞行时间质谱分离鉴定中国人乳中的β-酪蛋白[J]. 色谱, 2013, 31(5): 429-434.

HUANG Yu, REN Haowei, LIU Biao, LIU Ning, LI Meng, WANG Dongmao. Separation and identification of β-casein from Chinese human milk by ion exchange chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry[J]. Chinese Journal of Chromatography, 2013, 31(5): 429-434.

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离子交换色谱-基质辅助激光解吸电离串联飞行时间质谱分离鉴定中国人乳中的β-酪蛋白

Separation and identification of β-casein from Chinese human milk by ion exchange chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry

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