lc-msms法对融合蛋白fp3的氨基酸全序列测定.pdfVIP

216 药学学报 Acta Pharmaceutica Sinica 2012, 47 (2) : 2 16 222 LC-MS/MS 法对融合蛋白FP3 的氨基酸全序列测定 1 2 1 1 1 1* 1* 响 , 高向东 , 陶 磊 , 裴德宁 , 郭 莹 , 饶春明 , 王军志 ( 1. 中国食品药品检定研究院重组技术产品室, 卫生部生物技术产品检定方法及其标准化重点实验室, 北京 100050; 2. 中国药科大学生命科学与技术学院, 江苏 南京 210009) 摘要: 运用液质联用、两种串联质谱对融合蛋白FP3 的氨基酸全序列测定, 确证其一级结构。将样品还原烷 基化后, 通过胰蛋白酶酶解蛋白, PNGase F 去除多肽混合物中糖肽的糖基化, 将去糖后的总肽用于液质联用系统, 通过液相分离后, 运用Q-TOF 和线性离子阱两种串联质谱测定各个肽段的b, y 碎片离子, 分析测定融合蛋白FP3 的氨基酸全序列。通过LC-ESI-Q-TOF 完成了融合蛋白FP3 的76%氨基酸序列测定, 通过LC-ESI-Trap 完成余下 24%氨基酸序列测定。液质联用、串联质谱法测定蛋白质氨基酸序列快速、灵敏、准确, 是对重组蛋白结构分析 确证的重要手段。 关键词: 液质联用; 融合蛋白; 氨基酸序列 中图分类号: R917 文献标识码:A 文章编号: 05 13-4870 (2012) 02-02 16-07 Measurement of the amino acid sequence for the fusion protein FP3 with LC-MS/MS 1 2 1 1 1 1* 1* LI Xiang , GAO Xiang-dong , TAO Lei , PEI De-ning , GUO Ying , RAO Chun-ming , WANG Jun-zhi (1. Dep artment of Recombinant Product, National Institutes for Food and Drug Control, Key Laboratory of t he M inistry of Health for Research on Quality and Standardization of Biotech Products, Beij ing 100050, China; 2. School of Life Science and Technology, China Pharmaceutical University, Nanj ing 210009, China) Abstract: The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The m