Citation: LI Xianyu, ZHAO Xinyuan, YING Wantao, QIAN Xiaohong. Classification and identification of the secretome of hepatocellular carcinoma cells by isoelectric focusing electrophoresis pre-fractionation strategy[J]. Chinese Journal of Chromatography, ;2013, 31(9): 831-837. doi: 10.3724/SP.J.1123.2013.02010 shu

Classification and identification of the secretome of hepatocellular carcinoma cells by isoelectric focusing electrophoresis pre-fractionation strategy

LI Xianyu ^1,2 ,
ZHAO Xinyuan ^1,2 ,
YING Wantao ^2,, ,
QIAN Xiaohong ^1,2,,

1.
1. College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100022, China;

Corresponding author: YING Wantao, QIAN Xiaohong,
Received Date: 6 February 2013
Available Online: 20 March 2013
Fund Project: 国家重点基础研究"973项目"(2011CB910603) (2011CB910603)国家高技术研究发展计划项目(2012AA020203) (2012AA020203)北京市科技新星计划项目(Z121107002512014). (Z121107002512014)

Secretome (secreted proteins) has been arousing considerable attention because of its potential to develop novel biomarkers for the early detection and diagnosis of diseases. Like other proteome systems, a secretome generally consists of thousands of proteins with a wide dynamic range of abundance. The effective way to decrease the complexity of the analyte is to combine different prefractionation methods with orthogonal separation capabilities. The newly appeared OFFGEL fractionation system provides the ability to separate the peptides into discrete liquid fractions according to their differences in isoelectric point (pI), and has demonstrated high efficiency and repeatability for the analysis of the complex proteins. Here the OFFGEL fractionation system was evaluated for the separation of peptides digests from secreted proteins of hepatocellular carcinoma cells (HCC), using a 12-wells device encompassing the pH range of 3-10. In combination with the online reversed phase liquid chromatography and mass spectrometry, peptides from the digestion of MHCC97L secreted proteins were separated and identified. Preliminary results showed that nearly 80% of the peptides identified were specific to one fraction, and 40% of the proteins were obtained from the fractions 1-3 from the acid end. The average of peptide pI values distributed in each fraction is consistent with the theoretical pIs, and the pI distribution of the peptides focused on the low pH range showed a smaller deviation. Finally, 2995 proteins were identified from secretome of MHCC97L cell lines. Thus the data show that the OFFGEL system provides a highly valuable tool to fractionate peptides from complex protein samples.
- isoelectric focusing,
- pre-fractionation,
- secretome,
- classification,
- identification,
- hepatocellular carcinoma cells (HCC)
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