An off-line high pH reversed-phase fractionation and nano-liquid chromatography-mass spectrometry method for global proteomic profiling of cell lines

doi:10.1016/j.jchromb.2014.10.031

CORC > 上海药物研究所 > 中国科学院上海药物研究所 > 化学蛋白质组学研究中心

	An off-line high pH reversed-phase fractionation and nano-liquid chromatography-mass spectrometry method for global proteomic profiling of cell lines
	Wang, Hang 1; Sun, Shengnan 2; Zhang, Yi 2; Chen, Si 1; Liu, Ping 2; Liu, Bin 2
刊名	JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
	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.
资助项目	National Natural Science Foundation of China[21405104]
WOS关键词	HYDROPHILIC INTERACTION CHROMATOGRAPHY ; STRONG-CATION-EXCHANGE ; RAT-KIDNEY PROTEOME ; MULTIDIMENSIONAL SEPARATION ; SHOTGUN PROTEOMICS ; PEPTIDE SEPARATION ; PEAK-CAPACITY ; PROTEINS ; ELECTROPHORESIS ; PHOSPHOPROTEOME
WOS研究方向	Biochemistry & Molecular Biology ; Chemistry
语种	英语
出版者	ELSEVIER SCIENCE BV
WOS记录号	WOS:000346540800012
内容类型	期刊论文
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.