Mass spectrometrical identification of brain proteins including highly insoluble and transmembrane proteins
Mass spectrometrical identification of brain proteins including highly insoluble and transmembrane proteins
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DOI：
10.1016/j.neuint.2006.01.013
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发表时间：
2006-08-01
影响因子：
4.2
通讯作者：
Lubec, Gert
中科院分区：
文献类型：
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作者：
Bierczynska-Krzysik, Anna;Kang, Sung Ung;Lubec, Gert
传统的双向电泳（2DE）是用于组织和细胞蛋白质谱分析的主要技术，然而，强酸性、碱性或高度不溶性蛋白质的分离仍然是有限的。已经提出了一系列的方法来科普这个问题，并使用不连续凝胶电泳在酸性缓冲液系统中使用阳离子洗涤剂苄基二甲基-正十六烷基氯化铵（16-BAC），随后的SDS-PAGE，然后通过质谱表明，从2DE的结果可以补充这种方法。本研究的目的是从小鼠全脑中分离和鉴定2DE不能证明的蛋白质。为此，将样品均质化，通过超离心除去可溶性蛋白质，并将水不溶性沉淀物重悬于含有尿素、16-BAC、甘油、派洛宁Y和二硫苏糖醇的混合物中。在16-BAC存在下进行电泳，从含有分离的蛋白质的凝胶上切下条带，并在SDS-PAGE上再次进行电泳。通过MALDI-TOF-TOF质谱分析蛋白质点，共鉴定出187个蛋白质点，其中pI > 8.0的42个，pI <6.0的25个，pI < 6.0的39个。共鉴定出12个具有跨膜结构域（1 ~ 8个）的蛋白，包括通道和载体。生成的图谱揭示了一系列以前没有分离和鉴定的重要脑蛋白。因此，该系统可能与通道和载体的蛋白质化学测定相关，而与抗体的可用性和特异性无关。具有阳性肉汁指数的跨膜、碱性、酸性以及疏水性蛋白质可以被解析的事实保证了进一步改进该分析工具的工作。(c)2006爱思唯尔有限公司保留所有权利。
Conventional two-dimensional electrophoresis (2DE) is the main technique used for protein profiling of tissues and cells, however separation of strongly acidic, basic or highly insoluble proteins is still limited. A series of methods have been proposed to cope with this problem and the use of discontinuous gel electrophoresis in an acidic buffer system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC) with subsequent SDS-PAGE followed by mass spectrometry showed that results from 2DE can be complemented by this approach. It was the aim of this study to separate and identify proteins from whole mouse brain that were not demonstrated by 2DE. For this purpose samples were homogenised, soluble proteins were removed by ultracentrifugation and the water-insoluble pellet was resuspended in a mixture containing urea, 16-BAC, glycerol, pyronine Y and dithiothreitol. Electrophoresis was run in the presence of 16-BAC, the strip from the gel containing separated proteins was cut out and was re-run on SDS-PAGE. Protein spots were analyzed by MALDI-TOF-TOF mass spectrometry.One hundred and six individual proteins represented by 187 spots were unambiguously identified consisting of 42 proteins with predicted pI values of pI > 8.0, 25 with a 6.0 < pI < 8.0 and 39 with a pI < 6.0. Twelve proteins with transmembrane domains (ranging from 1 to 8) including channels and carriers were identified. The generated map revealed a series of important brain proteins that were not separated and identified previously. Therefore, this system may be relevant for protein chemical determination of channels and carriers independent of antibody availability and specificity. The fact that transmembrane, basic, acidic as well as hydrophobic proteins with a positive Gravy Index can be resolved warrants work on further improvement of this analytical tool. (c) 2006 Elsevier Ltd. All rights reserved.