【作者】 陈静； 陈枢青；

【Author】 CHEN Jing CHEN Shu-Qing Department of Biopharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310031. P. R. China

【机构】 浙江大学药学院生物制药研究室；

【摘要】 利用先进的生物质谱技术蚋升电喷雾串联质谱鉴定从大肠杆菌中表达酶切加工后得到 PTH(1-34)样品．通过ESI-MS测定重组PTH(1-34)的分子量及ESI-MS/MS对其胰蛋白酶酶解产物进行肽质量指纹谱分析,获得各肽段的分子量信息．并对其中的四个肽段用串联质谱分析各个肽段,结合数据库查询获得其完整的一级结构信息．PTH(1-34)测定分子量为4115．21,与理论值相比测定相差0．06％．ESI-MS/MS测定出其中双电荷离子峰m/z 728．4肽段序列为(S) VSEIQLMHNLG(K),以及其他三个单电荷离子峰的序列．查询数据库后确定重组表达的PTH(1-34) 一级结构正确无误,这说明在多肽的重组表达,纯化酶切的加工过程,没有出现氨基酸的丢失、突变和修饰．蚋升电喷雾串联质谱以其灵敏、快速和准确为重组蛋白质鉴定提供了有效的手段．更多还原

【Abstract】 Recombinant human PTH(l-34) was identified by new technique nanoESI- TOF tandem mass spectrometry. The calculated molecular weight of PTH(l-34) was 4117.76 by ESI-MS and the measured value was 4115.21. The measurement percent error was only 0.06 %. Digested PTH(1-34) by trypsin at 37℃ over night The tryptic digested peptides were measured and then four charged peptides were selected to measure their amino acid sequence by ESI-MS/MS. Through analysis the a, b. y series ions, the sequence of peptide m/z 728.4 measured by ESI-MS/MS was VSEIQLMHNLGK and then the database search results with other three peptides were both PTH(1-34). The study proves that the primary structure of PTH(1-34) is correct and there is no amino acid deletion, mutation and modification in the process of expression , purification and digestion. It indicate that nanoESI-MS/MS is a good method to identify protein with advantage of sensitivity, high speed and accuracy.更多还原