等电聚焦预分离用于肝癌细胞分泌蛋白质组的分级与鉴定

分泌蛋白质组(secretome)是指在特定的时空条件下,细胞、组织等分泌的全部蛋白质。分泌蛋白质组可能包含了大量的疾病诊断生物标志物,因此其相关研究越来越受到重视。分泌蛋白质组的组成高度复杂且浓度范围宽,这对分析方法提出了挑战。建立有效的蛋白质或肽段预分离策略,将有利于分泌蛋白质的高覆盖率鉴定。本研究以肝癌细胞系MHCC97L的无血清培养分泌蛋白质为研究对象,采用一种新型等电聚焦预分离(OFFGEL)系统,考察了肽段水平的分级对蛋白质鉴定结果的影响。结果表明,分离后各馏分中肽段的等电点分布与理论预测基本一致,每个馏分中单独鉴定的肽段比例接近80%,显示了该系统对肽段的高分辨分离能力。结合生物质谱技术,在肝癌细胞分泌系统中鉴定了2995个蛋白质,显示了该系统在复杂体系蛋白质组研究中的应用潜力。

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

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.