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Electrophoretic Separation of Proteins
Published on: June 12, 2008
用电子捕获解离质谱法对较大的蛋白质 (45 kDa) 进行自上而下的表征
Ying Ge1, Brian G Lawhorn, Mariam ElNaggar
1Baker Laboratory, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Journal of the American Chemical Society
|January 24, 2002
概括
以电子捕获解离 (ECD) 的自上而下的质谱法可以进行详细的蛋白质结构特征. 这种方法成功地识别和分析了参与胺生物合成,辅酶A合成和病毒prolyl-4-hydroxylase的蛋白质,揭示了翻译后的修改.
科学领域:
- 蛋白质组学和质谱学
- 蛋白质的结构特征 蛋白质的结构特征
- 生物化学 生物化学
背景情况:
- 从基因组数据中确定蛋白质的结构是蛋白质组学中的一个重大挑战.
- 准确的蛋白质表征需要分离,序列识别和修改分析.
- 像自下而上的蛋白质分解这样的现有方法在全面的蛋白质分析中存在局限性.
研究的目的:
- 应用和评估由电子捕获解离 (ECD) 增强的自上而下的质谱 (MS) 方法,用于详细的蛋白质结构特征.
- 为了研究涉及胺生物合成的蛋白质,辅酶A生物合成和病毒prolyl-4-hydroxylase.
- 在复杂蛋白质混合物中识别测序错误和翻译后修改.
主要方法:
- 利用"自上而下"的富里叶变换质谱法 (FTMS) 与电喷射电离 (ESI) 分析复杂的大肠杆菌细胞提取物.
- 雇佣的MS/MS和电子捕获解离 (ECD) 用于增强蛋白质碎片化和结构分析.
- 将自上而下的FTMS/ECD与传统的"自下而上的"蛋白质分解方法进行比较.
主要成果:
- 鉴定了来自胺生物合成的ThiS和ThiG蛋白质,揭示了N-终端修饰 (ThiS上的额外Met,ThiG上的Met和Ser去除).
- 来自辅酶A生物合成的特征性合酶/脱碳酶 (CoaBC),识别N终端Met去除.
- 分析了病毒的prolyl-4-hydroxylase (P4H),检测了酸添加物,并证实了Met去除和二硫化键形成.
- 对这些复杂蛋白质来说,自上而下FTMS/ECD的表现优于自下而上蛋白解.
结论:
- 上向FTMS与ECD相结合,显著提高了大型蛋白质详细结构表征的能力.
- 该方法准确地识别了翻译后的修改和序列变化,克服了其他方法的局限性.
- 这种综合方法对于推进蛋白质组学和了解蛋白质功能至关重要.
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