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学科主题物理化学
Integration of Cell Lysis, Protein Extraction, and Digestion into One Step for Ultrafast Sample Preparation for Phosphoproteome Analysis
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; Zou HF(邹汉法); Ye ML(叶明亮)
刊名ANALYTICAL CHEMISTRY
2014-07-15
ISSN0003-2700
DOI10.1021/ac5002146
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
资助者1,1 ; 1,1 ; 1,1 ; 1,1
类目[WOS]Chemistry, Analytical
研究领域[WOS]Chemistry
关键词[WOS]IMMOBILIZED TRYPSIN DIGESTION ; INTENSITY FOCUSED ULTRASOUND ; PROTEOMICS ; CHROMATOGRAPHY ; IMPACT ; MASS
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.
语种英语
资助者1,1 ; 1,1 ; 1,1 ; 1,1
原文出处查看原文
WOS记录号WOS:000339227400010
引用统计
被引频次:12[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/119725
专题中国科学院大连化学物理研究所
通讯作者Zou HF(邹汉法); Ye ML(叶明亮)
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
推荐引用方式
GB/T 7714
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.
APA Liu, Fangjie.,Ye, Mingliang.,Pan, Yanbo.,Zhang, Yi.,Bian, Yangyang.,...&叶明亮.(2014).Integration of Cell Lysis, Protein Extraction, and Digestion into One Step for Ultrafast Sample Preparation for Phosphoproteome Analysis.ANALYTICAL CHEMISTRY,86(14),6786-6791.
MLA Liu, Fangjie,et al."Integration of Cell Lysis, Protein Extraction, and Digestion into One Step for Ultrafast Sample Preparation for Phosphoproteome Analysis".ANALYTICAL CHEMISTRY 86.14(2014):6786-6791.
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