基于亲和色谱的肺癌细胞磷酸化蛋白质组研究及其应用  

Affinity chromatography based phosphoproteome research on lung cancer cells and its application

作　　者：张宝会 王晨桐 郭淼 肖华[1] ZHANG Baohui;WANG Chentong;GUO Miao;XIAO Hua(State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China)

机构地区：[1]微生物代谢国家重点实验室,上海交通大学生命科学技术学院,上海200240

出　　处：《色谱》2021年第1期77-86,共10页Chinese Journal of Chromatography

基　　金：国家重点研发计划(2017YFC1200204);国家自然科学基金(21675110).

摘　　要：磷酸化是蛋白质翻译后修饰的重要形式之一,其异常往往会导致细胞内信号通路的紊乱和疾病的发生。固定化金属离子亲和色谱(IMAC)是磷酸化肽段的高效富集技术,在磷酸化蛋白质组研究方面应用广泛。该研究以金属钛离子(Ti^4+)螯合IMAC材料(Ti^4+-IMAC)为载体,进行磷酸化肽段富集。比较了10μm Ti^4+-IMAC通过振荡法和固相萃取法(SPE)富集磷酸肽的效果,发现振荡法可以富集到更多的磷酸肽;对比了两种尺寸(10μm和30μm)Ti^4+-IMAC在磷酸化肽段富集中的差异,发现小尺寸材料富集效果更佳。进一步采用优化的策略比较了不同转移能力肺癌细胞的磷酸化蛋白质组,免标记定量蛋白质组学结果表明,优化的Ti^4+-IMAC方法可以从正常的肺成纤维细胞MRC5、低转移肺癌细胞95C和高转移肺癌细胞95D中分别鉴定到510、863和1108种磷酸化蛋白质,其中317种为3组所共有。该研究共鉴定到1268种磷酸化蛋白质上的7560个磷酸化位点,其中1130个为差异磷酸化位点,文献报道显示部分异常表达的激酶与癌症转移密切相关。通过生信对比分析发现,异常表达的磷酸化蛋白质主要与细胞侵袭、迁移和死亡等细胞迁移方面的功能有关。通过优化磷酸化肽富集策略,初步阐明了磷酸化蛋白质网络的异常与肺癌转移之间的相关性,该方法有望用于肺癌进展相关的磷酸化位点、磷酸化蛋白质及其信号通路研究。Phosphorylation is one of the most important post-translational modifications in proteins.It plays a key role in numerous cellular processes,including signal transduction,cell proliferation,and intercellular communication.More than 30%of the cellular proteins are phosphorylated at a given time.However,dysregulation of phosphorylated proteins usually leads to a disorder in the intracellular signaling pathways and the onset of various diseases,especially cancer.Cell proliferation and metastasis are the major manifestations of cancer progression,and these might be affected by the protein phosphorylation levels.Clinically,cancer usually metastasizes at the middle and late stages,affecting other organs beyond primary lesion.This poses significant challenges in cancer treatment and prognosis.Consequently,comparing the phosphorylated proteomes of cells with different metastatic capabilities is helpful in studying the role of protein phosphorylation in cancer metastasis and progression.The human low metastatic lung cancer cell line 95C and high metastatic lung cancer cell line 95D are two of the four sublines isolated from human lung giant cell carcinoma cell line(PLA-801)by the single-cell cloning technique.These are ideal models for studying tumor metastasis and non-small cell lung cancer.MRC-5 cell line was obtained from a 14 week old fetal normal lung tissue.Quantitative analysis of the proteome and phosphorylated proteome in these normal lung cells and lung cancer cells with different metastatic capacities can identify key pathways and regulatory proteins associated with lung cancer metastasis and progression.Immobilized metal affinity chromatography(IMAC)is an efficient technique for the enrichment of phosphopeptides and has been widely used for phosphoproteome research.Metal ions(such as Ti^4+)are immobilized on the substrate by chelation,and phosphopeptides can be selectively adsorbed under acidic conditions and eluted under alkaline conditions.IMAC can enrich phosphate groups at different amino acid sites with h

关 键 词：固定化金属离子亲和色谱 磷酸化 蛋白质组 癌症转移 肺癌细胞 

分 类 号：O658[理学—分析化学]