集成化蛋白质组定量分析平台的构建及应用

Establishment and application of integrated platform for proteome quantification

为提高蛋白质组定量分析的准确度、通量和自动化程度,构建了由微升级混合离子交换色谱、亲水型固定化酶反应器(hIMER)和纳升级反相色谱-电喷雾串级质谱(nanoRPLC-ESI-MS/MS)组成的集成化蛋白质定量分析平台。该平台实现了二甲基化标记蛋白质样品在线分离、酶解、肽段分离鉴定和定量分析。采用质量比为1∶1的轻、重标记的蛋白质样品考察该平台的定量性能,发现蛋白质水平二甲基化标记效率为90%;蛋白质经hIMER在线酶解10 min产生的漏切及酶解产物在hIMER柱上的非特异性吸附对定量准确度的影响较小,所有定量到的重/轻标记的蛋白质质量比的平均值为1.01。最后将该平台应用于小鼠腹水型肝癌淋巴道高、低转移细胞系差异蛋白质的分析,发现了12种蛋白质在高转移细胞系中低表达,15种蛋白质在高转移细胞系中高表达。以上结果证明了该平台可以实现高准确度和高通量的蛋白质组定量分析。

To improve the accuracy,throughput and automation of proteome quantification analysis,an integrated platform including a microflow mixed-bed ion exchange column,a hydrophilic immobilized enzymatic reactor （ hIMER ） and nanoRPLC-electrospray ionization （ESI）-MS / MS system was established. Online separation and digestion of dimethylated pro-teins,followed by peptide separation,identification and quantification can be realized automat-ically by this platform. High and light dimethyl-labeled（H / L）proteins with the mass ratio of 1 ：1 were used to evaluate the quantification performance of the platform. The results showed that the dimethyl labeling efficiency at protein level was 90% . The incomplete digestion resulting from 10 min online digestion by the hIMER column and the non-specific adsorption of protein digests on the column had little adverse effect on the accuracy of protein quantification results. The mean value of H / L（mass ratio）of all the quantified proteins was 1. 01. This platform was finally applied to analyze the different protein expression levels of two mice hepatocarcinoma ascites cell lines with high and low lymph node metastasis rates（Hca-F and Hca-P cell lines）. Finally 15 up-regulated and 12 down-regulated proteins （ Hca-F / Hca-P ） were successfully obtained. All these results demonstrated that the integrated platform can be used for proteome quantification with the advantages of high accuracy and high throughput.