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多磷酸蛋白对于生物体适应内外环境具有重要意义,而明确多磷酸蛋白的磷酸位点功能及其信号转导机制尤为关键.复杂生物样品中多磷酸化肽的低丰度、低电离的特性,以及非磷酸化肽的抑制作用,决定了质谱分析前进行多磷酸化肽富集是非常必要的步骤.本工作采用基于巯基-烯烃点击化学法合成的混合模式材料Click TE-GSH进行单磷酸化肽和多磷酸化肽的选择性富集.我们建立了单磷酸化肽、双磷酸化肽和多磷酸化肽的顺序分段富集方法.该优化方法能抗干扰,应用于脱脂牛奶时富集到11条多磷酸化肽.与商品化固化金属亲和色谱(IMAC)材料相比,Click TE-GSH富集多磷酸化肽的选择性更好.本工作所建立的富集方法为高效富集多磷酸化肽提供新方法和新技术.
Phosphorylation of polyphosphates is of great importance for the organism to adapt to the internal and external environment, and it is particularly crucial to clarify the function of the phosphorylation site of polyphosphoric acid protein and its signal transduction mechanism.Low abundance and low ionization of polyphosphorylated peptides in complex biological samples, As well as the inhibition of non-phosphorylated peptides, which determine the enrichment of polyphosphorylated peptides before mass spectrometry is a necessary step.In this work, we performed single phosphorylation using Click TE-GSH, a hybrid mode material based on thiol-olefin click chemistry Peptide and polyphosphorylated peptides were selectively enriched.We established sequential fragment enrichment methods for monophosphorylated peptide, bisphosphorylated peptide and polyphosphorylated peptide.This optimization method is anti-interference, Aggregated to 11 polyphosphorylated peptides.The Click TE-GSH-enriched polyphosphorylated peptide was more selective than the commercially available immobilized metal affinity chromatography (IMAC) material.The enrichment method established in this work was highly efficient Enrichment of polyphosphorylated peptides provides new methods and new technologies.