基于 LC Chip Q-TOF MS/MS 的非标记比较策略研究丙二酰黄芪皂苷 I 抗白血病 U937 细胞的差异蛋白质组(英文)  

Proteomic Analysis for Malonylastragaloside I in U937 Leukemia Cells by Modified Label-free Quantitative Strategy with LC Chip Q-TOF MS/MS

作　　者：彭咏波[1] 周萍[1] 楚楚[1] 刘鄂湖[1] 闻晓东[1] 刘群[1] 陈君[1] 高宁[2] 齐炼文[1,3] 李萍[1,3] 

机构地区：[1]中国药科大学天然药物活性物质与功能国家重点实验室,南京210009 [2]中国人民解放军第三军医大学药学院,重庆400038 [3]中国药科大学生药学教研室,南京210009

出　　处：《中国天然药物》2011年第4期305-316,共12页

基　　金：supported by Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0868);National Science and Technology Major Project 'Creation of Major New Drugs' from China (No. 2009ZX09502-020)~~

摘　　要：目的:研究丙二酰黄芪皂苷 I 抗白血病 U937 细胞增殖的差异表达蛋白质组。方法:基于 LC Chip Q-TOF 系统建立和优化了一种非标记蛋白质组比较方法-多肽色谱峰面积。方法学研究表明,该方法操作简单、方便,具有较高的重复性和宽动力学范围。然后,将建立的优化方法组合归一化计算应用于新化合物丙二酰黄芪皂苷 I 抗白血病 U937 细胞增殖的差异蛋白质组表达研究。结果:分析结果表明,2 倍以上表达差异的蛋白质 15 个,其中 6 个蛋白质的表达差异获得了 Western blotting 确证。此外,通过差异蛋白质的相互作用分析,PARP1、Caspase-3 和-9 的 Western blot 检测及 Caspase 抑制剂实验,我们发现 Caspase激活在丙二酰黄芪皂苷 I 抗白血病 U937 细胞增殖效应中起重要作用。结论:通过该优化的非标记蛋白质组比较方法,找到了一些与丙二酰黄芪皂苷 I 抗白血病 U937 细胞增殖密切相关的功能蛋白质,为深入开展其分子机理研究奠定了基础。AIM：To study the differentially-expressed proteins for malonylastragaloside I in human leukemia U937 cells.METHODS：A suitable protein quantitative mode of label-free technique was established by LC Chip Q-TOF.It was found that the mode of peptide spectral intensity based on label-free quantification strategy of LC Chip Q-TOF showed high reproducibility,wide dynamic range,as well as simplicity and convenience.The optimized quantification mode of the peptide spectral intensity,normalization algorithm was applied to screen the differentially-expressed proteins for malonylastragaloside I（MA-I）,a novel astragaloside from Astragalus sp.,in human leukemia U937 cells.RESULTS：Fifteen differentially-expressed proteins with up or down-regulation over 2.0 folds were identified,six of which including HSC70,RPLP2,PHB2,PCNA,TCTP and ANXA11 were confirmed by Western blotting.We also observed significant activation of PARP1 and cleavage of caspases-3 and-9 by MA-I in U937 cells,and the pan-caspase inhibitor Z-VAD-FMK could reduce MA-I inhibited cell proliferation,indicating that caspase activation may be involved in MA-I induced cell toxicity.CONCLUSION：This study has generated potentially functional proteins important for MA-I inhibited U937 cells proliferation by the optimized quantification mode of peptide spectral intensity.

关 键 词：LC CHIP Q-TOF 非标记 多肽色谱峰面积 丙二酰黄芪皂苷 I 细胞增殖 

分 类 号：Q71[生物学—分子生物学]