人健康肝组织核心岩藻糖基化蛋白质的鉴定及生物信息学分析  

Characterization of Core-fucosylated Glycoproteins From Human Normal Liver Tissues

作　　者：代智[1] 樊嘉[1,2] 周俭[1] 柏斗胜[1] 邱双健[1] 康晓楠[2] 崔杰峰[1] 郭坤[1] 李岩[1] 刘银坤[1,2] 

机构地区：[1]复旦大学附属中山医院肝癌研究所,教育部癌变和侵袭原理重点实验室,上海200032 [2]复旦大学生物医学研究院,上海200032

出　　处：《生物化学与生物物理进展》2008年第5期555-562,共8页Progress In Biochemistry and Biophysics

基　　金：国家高技术研究发展计划资助项目(863)(2007AA02Z479);上海市医学领军人才资助项目(LJ06004);高等学校博士学科点专项科研基金资助项目(20070246112);复旦大学青年科学基金资助项目(EYF152008)~~

摘　　要：糖蛋白质组学方法鉴定人健康肝组织核心岩藻糖基化蛋白质,将有助于发现肝癌、慢性肝病相关异常核心岩藻糖基化蛋白质.应用凝集素亲和层析技术、双向电泳(2-DE)联合基质辅助激光解吸飞行时间串联质谱(MALDI-TOF-MS/MS)分析,建立人健康肝组织核心岩藻糖基化蛋白表达谱,图谱均点数为(130±3)个,挖取90个蛋白质点进行质谱鉴定,数据库搜索及去冗余后,共鉴定53种蛋白质,主要分布于pI5～9,分子质量为10～100ku区域处.Gene Ontology分类发现它们参与体内代谢等过程,应用NetNGlyc对它们进行糖基化位点预测,并通过蛋白质免疫沉淀联合凝集素亲和印迹对其中的结合珠蛋白前体,α烯醇化酶的核心岩藻糖基化进行了再验证.以上结果提示,凝集素层析、2-DE联合MALDI-TOF-MS/MS分析是一种鉴定某种特定类型糖蛋白的高通量检测方法,所建立的表达谱可用于发现疾病发生、发展中相关的异常核心岩藻糖基化蛋白质.In order to establish and analyze core-fucosylated glycoprotein profiles of human normal liver tissues, which could contribute to the finding of more aberrantly fucosylated glycoproteins related to liver diseases, based on the approach, ＂lectin affinity chromatography, 2-DE and MALDI-MS/MS＂ , the lens culinaris agglutinin （LCA） affinity glycoprotein profiles from human normal liver tissues were obtained, in which 130±3 protein spots were detected. Altogether, 90 silver-stained spots in the 2-DE map were cut out, destained and subjected to in-gel tryptic digestion, and 53 proteins were identified by MALDI-TOF MS/MS. These proteins, mainly in the middle range （pI 5 -9, M 10- 100 ku）, were found to participate in the metabolism and play crucial roles in binding and catalytic reactions. Putative N-linked glycosylation consensus sequence, i.e., -N-X-S/T-, was applied to evaluate glycosylation of all the identified proteins. Furthermore, protein immunoprecipitation combined with lectin blot was used to further validate fucosylation of the identified possible candidate proteins, haptoglobin and alpha enolase. All the results suggested that lectin affinity chromatography and 2-DE in combination with MALDI-MS/MS enabled the identification of all the specific subsets of glycoprotein, and the database could give us a help when searching for some aberrantly core-fucosylated glycoproteins associated with some diseases.

关 键 词：核心岩藻糖蛋白 扁豆凝集素 亲和层析 糖蛋白质组学 人健康肝组织 

分 类 号：Q513.2[生物学—生物化学] R333.4[医药卫生—人体生理学]