基于双功能荧光标签8-氨基1,3,6-萘三磺酸的N-连接糖基化富集方法

Exploring the N-linked glycosylation capture method based on bio-functional fluorescent tag 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt

发展了基于双功能荧光标签8-氨基1,3,6-萘三磺酸(ANTS)选择性糖基化蛋白质标记和二氧化钛(TiO2)亲和富集的N-连接糖基化富集新方法.模式蛋白质细胞色素c、肌红蛋白、核糖核酸酶b、抗生素和α-1-抗胰蛋白酶经ANTS标记后,所有糖基化蛋白质均发出亮绿色荧光,而非糖基化蛋白质则不能观测到荧光;进一步采用TiO2对酶切产生的N-连接糖基化肽段进行富集,同时实现了中性糖肽和唾液酸糖肽的选择性富集.通过进一步分析人血清N-连接糖基化蛋白质,共鉴定到39个唯一的N-连接糖蛋白,包括了53个唯一的N-连接糖基化位点;此外,该方法与采用TiO2法直接富集以及肼化学法富集鉴定到的交叠糖基化位点数均小于40%,说明这一新方法与传统方法在糖基化肽段富集方面具有一定的互补性,有望用于复杂蛋白质样品的N-连接糖基化高效富集.

The method with the combination of bio-functional fluorescent tag 8-aminonaphthalene-1,3, 6-trisulfonic acid （ANTS） based N-linked glycoprotein labeling and titanium dioxide （TiO2） for N-linked glycopeptide enrichment was developed. With proteins cytochrome c, myoglobin, ribonuclease b, avidin, and alpha-1-antitrypsin as the samples, all of the glycoproteins were selectively labeled, emitting strong green fluorescence, while the non-glycoproteins had no fluorescence emission at all. With further capture by （TiO2）, both of the sialic acid glycopeptides and neutral glycopeptides were efficiently enriched. By further analyzing of N-linked glycoproteins from human serum, totally 39 unique N-linked glycoproteins including 53 unique N-glycosites were successfully identified. In comparison to direct capturing by TiOzand traditional hydraizde chemistry strategy, an overlap of less than 40% for N-linked glycosites was a- chieved, indicating the complementary nature of this protocol with directly TiO2 enrichment or traditional hydrazide chemistry. All these results suggested the potential merits of the new method for the high effi-cient N-linked glycosylation enrichment.