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学科主题: 物理化学
题名: Integration of Cell Lysis, Protein Extraction, and Digestion into One Step for Ultrafast Sample Preparation for Phosphoproteome Analysis
作者: Liu, Fangjie1, 2;  Ye, Mingliang1;  Pan, Yanbo1, 2;  Zhang, Yi1, 2;  Bian, Yangyang1, 2;  Sun, Zhen1, 2;  Zhu, Jun1, 2;  Cheng, Kai1, 2;  Zou, Hanfa1
通讯作者: 邹汉法 ;  叶明亮
刊名: ANALYTICAL CHEMISTRY
发表日期: 2014-07-15
DOI: 10.1021/ac5002146
卷: 86, 期:14, 页:6786-6791
收录类别: SCI
合作性质: 
文章类型: Article
部门归属: 18
项目归属: 1809
产权排名: 待补充
WOS标题词: Science & Technology ;  Physical Sciences
类目[WOS]: Chemistry, Analytical
资助者: 1,1
研究领域[WOS]: Chemistry
英文摘要: Conventional sample preparation protocols for phosphoproteome analysis require multiple time-consuming and labor-intensive steps, including cell lysis, protein extraction, protein digestion, and phosphopeptide enrichment. In this study, we found that the presence of a large amount of trypsin in the sample did not interfere with phosphopeptide enrichment and subsequent LC-MS/MS analysis. Taking advantage of fast digestion achieved with high trypsin-to-protein ratio, we developed a novel concurrent lysis-digestion method for phosphoproteome analysis. In this method, the harvested cells were first placed in a lysis buffer containing a huge amount of trypsin. After ultrasonication, the cells were lysed and the proteins were efficiently digested into peptides within one step. Thereafter, tryptic digest was subjected to phosphopeptide enrichment, in which unphosphorylated peptides, trypsin, and other components incompatible with LC-MS/MS analysis were removed. Compared with conventional methods, better phosphoproteome coverage was achieved in this new one-step method. Because protein solubilization and cell lysis were facilitated by fast protein digestion, the complete transformation of cell pellets into the peptide mixture could be finished within 25 min, while it would take at least 16 h for conventional methods. Hence, our method, which integrated cell lysis, protein extraction, and protein digestion into one step, is rapid and convenient. It is expected to have broad applications in phosphoproteomics analysis.
关键词[WOS]: IMMOBILIZED TRYPSIN DIGESTION ;  INTENSITY FOCUSED ULTRASOUND ;  PROTEOMICS ;  CHROMATOGRAPHY ;  IMPACT ;  MASS
语种: 英语
原文出处: 查看原文
WOS记录号: WOS:000339227400010
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/119725
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China

Recommended Citation:
Liu, Fangjie,Ye, Mingliang,Pan, Yanbo,et al. Integration of Cell Lysis, Protein Extraction, and Digestion into One Step for Ultrafast Sample Preparation for Phosphoproteome Analysis[J]. ANALYTICAL CHEMISTRY,2014,86(14):6786-6791.
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