| 一种适用于质谱分析的简化胶内酶解方法 |
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| 引用本文： | 王旭初 范鹏祥 李银心. 一种适用于质谱分析的简化胶内酶解方法[J]. 植物生理与分子生物学学报, 2007, 33(5): 449-455 |
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| 作者姓名： | 王旭初 范鹏祥 李银心 |
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| 作者单位： | 中国科学院植物研究所光合作用与环境分子生理学重点实验室，中国科学院植物研究所光合作用与环境分子生理学重点实验室，中国科学院植物研究所光合作用与环境分子生理学重点实验室 北京 100093，北京 100093:中国科学院研究生院，北京 100049，北京 100093 |
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| 基金项目： | 国家“863”计划项目(No．2007AA091705)，中国科学院重要方向性项目(No．KSCX2-YW-N-013)资助。~~ |
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| 摘 要： | 在常规方法的基础上,设计了一种适于质谱分析的简化胶内酶解方法。改进的步骤包括:(1)通过加大洗涤所用超纯水量、延长涡混时间来强化凝胶洗涤的环节;(2)加入酸化处理来提高胰蛋白酶的活性;(3)在预酶解时不加CaCl_2,减少了酶的自切作用;(4)省略了蛋白质样品脱盐、脱SDS的步骤;(5)直接吸取酶解液进行质谱分析。系统比较该简化酶解法和最新报道的一种酶解方法的质谱鉴定效果,简化法能有效减少酶解后肽段的损失,增加质谱数据库搜索的信息量,得到更可靠的蛋白质鉴定结果。
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| 关 键 词： | 蛋白质组 胶内酶解方法 双向电泳 肽质量指纹图谱 质谱分析 |
| 修稿时间： | 2007-04-24 |
A Simple Protein In-Gel Digest Method Compatible with Mass Spectrom- etry Anal3/sis |
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| WANG Xu-Chu, FAN Peng-Xiang, LI Yin-Xin Key Laboratory of Photosynthesis and Environmental Molecular Physiology. A Simple Protein In-Gel Digest Method Compatible with Mass Spectrom- etry Anal3/sis[J]. Journal Of Plant Physiology and Molecular Biology, 2007, 33(5): 449-455 |
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| Authors: | WANG Xu-Chu FAN Peng-Xiang LI Yin-Xin Key Laboratory of Photosynthesis Environmental Molecular Physiology |
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| Affiliation: | Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. |
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| Abstract: | A simple protein in-gel digest method compatible with MALDI-TOF MS analysis was developed from the previously reported protocols with the following modifications.(1)The wash- ing step was intensified by increasing the volume of distilled water and prolonging the vortex time. (2)Trypsin was acidified to improve the enzyme activity.(3)A Ca~(2 )free pre-digest solution was used to reduce the trypsin auto cleavage.(4)The procedure of removal of salts and SDS was cancelled.(5)The digest was directly used to per- form MALDI-TOF MS analysis.The compared results of this method with a latest reported proto- col demonstrated that the method could efficiently reduce the peptide loss,provide more information for MS analysis,and consequently make the pro- tein identification more reliable. |
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| Keywords: | proteomics in-gel digest method two-dimensional gel electrophoresis peptide mass fingerprinting mass spectrometry analysis |
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