多磷酸化肽标记技术
蛋白质磷酸化是生物界最普遍,也是最重要的一种蛋白质翻译后修饰,20世纪50年代以来一直被生物学家看作是一种动态的生物调节过程。在细胞中,大概有1/3的的蛋白质被认为是通过磷酸化修饰的。蛋白质的磷酸化修饰与多种生物学过程密切相关,如DNA损伤修复、转录调节、信号传导、细胞凋亡的调节等。磷酸化蛋白质及多肽的研究可以帮助人们阐述上述过程的机理,进一步认识生命活动的本质。近年来随着蛋白质组技术的不断发展,蛋白质磷酸化的研究越来越受到广泛的关注。
蛋白质磷酸化在细胞信号转导中的作用
磷酸化多肽主要指肽链中的Ser、Tyr和Thr残基的侧链羟基被修饰成酸式磷酸酯多肽。磷酸化多肽是研究蛋白质磷酸化过程的必不可少的工具,因此研究蛋白质及多肽的磷酸化反应并确定成熟简便的合成路线就变得非常重要。目前为止,多肽的磷酸化修饰主要有后磷酸化法和单体法两种合成方法。后磷酸化法是多肽序列在树脂上合成完后,再对其中的Ser、Tyr或Thr的侧链羟基进行磷酸化;单体法则是将适当保护的磷酸化氨基酸直接引入到多肽序列中,这种方法较后磷酸化法操作更为简便,已经成为多肽磷酸化修饰的主要方法。
单体法修饰时,磷酸化的氨基酸由于侧链修饰的较大基团产生的位阻而导致难以与肽链缩合,并且之后的氨基酸引入都会比较困难,尤其在含有多个磷酸化位点修饰时,合成将变得异常困难,并且最终产物成分复杂,难以分离,产率极低。因此,当肽链中多个位点进行磷酸化时,可以考虑采用后磷酸化法,其合成过程主要就是在多肽合成结束之后,选择性的脱去要标记氨基酸的侧链保护基,对于Tyr,Thr可以直接使用侧链不保护的氨基酸进行反应。侧链保护基在1%TFA/DCM条件下可以定量的脱除。后磷酸化时,可以采用双苄基亚磷酰胺,四氮唑生成亚磷酰胺四唑活性中间体,连接到羟基上,然后在过氧酸条件下氧化生成磷酰基,完成反应。
我们提供多肽进行两个,三个,四个,五个磷酸化位点修饰的高质量多肽。拥有成熟的合成纯化技术,不断进取的精英团队,国肽生物已经成为值得信赖的多肽供应品牌。
成功案例:
序列NH2-TERD(pS)D(pT)DVEEDSRPPGRPAEVHLERAQPFGFID(pS)D(pS)DAEEEY-CONH2,四个磷酸化修饰位点。
HPLC分析:
MS分析:
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