An off-line high pH reversed-phase fractionation and nano-liquid chromatography-mass spectrometry method for global proteomic profiling of cell lines
文献类型：期刊论文
| 作者 | Wang, Hang1; Sun, Shengnan2; Zhang, Yi2; Chen, Si1; Liu, Ping2; Liu, Bin2 |
| 刊名 | JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
 |
| 出版日期 | 2015 |
| 卷号 | 974页码:90-95 |
| 关键词 | Reversed-phase fractionation Nano-LC-MS/MS Off-line Global proteomics Cell lines |
| ISSN号 | 1570-0232 |
| DOI | 10.1016/j.jchromb.2014.10.031 |
| 文献子类 | Article |
| 英文摘要 | Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) and first-dimensional fractionation is widely used for reducing sample complexity in large-scale proteomic profiling experiments. However, the limited number of proteins identified and the relatively long running time are a barrier to the successful application of this approach. In this study, off-line high pH reversed-phase fractionation (RPF) was combined with nano-LC-MS/MS in order to develop an improved method for global proteomic profiling of different cell lines. In the first dimensional reverse phase HPLC separation, 300 mu g of digested cell protein was separated into 78 fractions under high pH conditions and condensed into 26 fractions for the second nano-LC-MS/MS analysis at low pH. The chromatographic conditions for the first and second steps were optimized, and the accuracy and reproducibility of protein quantification were investigated with an average Pearson correlation coefficient of 0.94. The method was then applied in the identification of proteins in six common cell lines (DMS, MFM, HepG2, U2OS, 293T and yeast), which resulted in identification of 7300-8500 and 8956 proteins in heavy/light labeled and label-free cell samples, respectively, in 1.5 days. The performance of the developed method was compared with isoelectric focusing (IEF)-nano-LC-MS/MS and the previously reported method; and off-line high pH RPF-nano-LC-MS/MS proved advantageous in terms of the number of proteins identified and the analytical time needed to achieve a successful global proteomic profiling outcome. The RPF-nano-LC-MS/MS method identified more proteins from low abundance (150 mu g) samples with an average sequence coverage for each cell line of 23.4-35.1%. RPF-nano-LC-MS/MS may therefore be an efficient alternative tool for achieving improved proteomic coverage of multiple cell lines. (C) 2014 Elsevier B.V. All rights reserved. |
| WOS关键词 | HYDROPHILIC INTERACTION CHROMATOGRAPHY ; STRONG-CATION-EXCHANGE ; RAT-KIDNEY PROTEOME ; MULTIDIMENSIONAL SEPARATION ; SHOTGUN PROTEOMICS ; PEPTIDE SEPARATION ; PEAK-CAPACITY ; PROTEINS ; ELECTROPHORESIS ; PHOSPHOPROTEOME |
| 资助项目 | National Natural Science Foundation of China[21405104] |
| WOS研究方向 | Biochemistry & Molecular Biology ; Chemistry |
| 语种 | 英语 |
| WOS记录号 | WOS:000346540800012 |
| 出版者 | ELSEVIER SCIENCE BV |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/276784]  |
| 专题 | 化学蛋白质组学研究中心 |
| 通讯作者 | Wang, Hang |
| 作者单位 | 1.Shanghai Jiao Tong Univ, Instrumental Anal Ctr, Shanghai 200240, Peoples R China; 2.Chinese Acad Sci, Shanghai Inst Mat Med, Beijing 201203, Peoples R China |
| 推荐引用方式 GB/T 7714 | Wang, Hang,Sun, Shengnan,Zhang, Yi,et al. An off-line high pH reversed-phase fractionation and nano-liquid chromatography-mass spectrometry method for global proteomic profiling of cell lines[J]. JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES,2015,974:90-95. |
| APA | Wang, Hang,Sun, Shengnan,Zhang, Yi,Chen, Si,Liu, Ping,&Liu, Bin.(2015).An off-line high pH reversed-phase fractionation and nano-liquid chromatography-mass spectrometry method for global proteomic profiling of cell lines.JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES,974,90-95. |
| MLA | Wang, Hang,et al."An off-line high pH reversed-phase fractionation and nano-liquid chromatography-mass spectrometry method for global proteomic profiling of cell lines".JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 974(2015):90-95. |
入库方式： OAI收割
来源：上海药物研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。