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酪氨酸激酶在生物分子的信号转导中起着非常重要的作用,目前除抗体技术外尚无有效的化学方法能够实现酪氨酸磷酸化蛋白质或多肽的选择性富集.然而抗体通常成本较高,而且往往会有模体序列的选择性识别.因此,本文发展了一种基于化学反应的酪氨酸磷酸化肽段的选择性富集方法.该方法利用了β-消除反应只能发生在丝氨酸和苏氨酸磷酸化多肽的特性,通过反相选择实现对酪氨酸磷酸化肽段的选择性富集.以标准多肽对其反应效率和回收率进行了考察,20 min内丝氨酸磷酸化多肽的β-消除反应效率可达99％以上,而同时酪氨酸磷酸化肽段可保持70％的回收率.进一步以6种标准蛋白质混合物的酶解产物对其进行考察,经β-消除反应和亲和富集之后,只有酪氨酸磷酸化多肽可以被检测出来.该方法为蛋白质酪氨酸磷酸化的分析提供了一种新的手段.

Tyrosine phosphorylation of proteins plays a vital role in signal transduction pathway.Currently,antibodybased method dominates the tyrosine phosphopeptide enrichment and there is a lack of other general approaches for selective isolation of tyrosine phosphopeptides.However,antibody-based methods are of high cost and biased to certain motifs.Here we developed a chemistry-based method for tyrosine phosphopeptide enrichment.This method utilized the β-elimination,which only occurs on phospho-serine/threonine residues,to achieve the reverse selection effect.After the dephosphorylation of serine/threonine phosphopeptides a sensitive phosphopeptide isolation method was applied to enrich tyrosine phosphopeptides.In this proof-of-concept study,it showed that the β-elimination for several standard serine phosphopeptides was completed over 99％ while the recovery of tyrosine phosphopeptide remained at around 70％ within 20 min.In the further test with 6-protein digests monitored by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis,only tyrosine phosphopeptides were detected.Due to the low-cost and unbiased properties,the method has potential applications in tyrosine signaling pathway analysis as an alternative to antibody-based approach.