胍基化修饰结合稀土金属标记应用于蛋白质相对定量研究

Improved Method for Protein Quantification by Using Rare Earth Metal-Chelated Tags after Guanidiation

稀土金属元素标记技术利用双功能试剂二乙三胺五乙酸双酸酐(DTPA双酸酐)将稀土金属元素和目标肽段连接起来作为质量标签,结合基质辅助激光解吸/电离-飞行时间质谱(MALDI-TOF MS)分析实现蛋白质的相对定量研究。为进一步提高标记效率,本研究对反应体系温度、pH值、反应时间和金属标签过量倍数等进行优化,并结合胍基化修饰技术封闭蛋白质酶切肽段中的侧链ε-氨基,使标记反应只发生在末端氨基,提高标记选择性。标准蛋白质实验结果表明,所有酶切肽段胍基化反应效率均大于95%,且胍基化修饰反应对金属标记效率没有影响。对于选定的7个标准蛋白质而言,每个蛋白质都至少有3个肽段可以得到良好的标记结果。对于离子抑制效应带来的干扰,可通过标准曲线进行校正,结果表明,当两种金属离子浓度比小于6时,该效应对蛋白质相对定量没有影响。胍基化修饰结合稀土金属标记方法不仅能在肽段水平实现相对定量,而且能在蛋白质水平实现相对定量。

Rare earth metal can be chelated to target peptides by using bicyclic anhydride diethylenetriamine-N,N,N＇,N＇＇,N＇＇＇-pentaacetic acid dianhydride（DTPA dianhydride）,which can be used as tags and analyzed by matrix-assisted laser desorption/ionization-time of flight MS（MALDI-TOF MS） for protein quantification.To further improve the labeling efficiency,reaction temperature,pH value,time and the amount of metal tag were optimized in this study.And for the first time,guanidiation was introduced to make the labeling only take place at the N-terminal of the peptide,thus the selectivity was improved.The results showed that the guanidiation rate of all peptides from standard proteins exceeds 95%,and guanidiation had little influence on the labeling.The results from seven standard proteins showed that at least three peptides of each protein can be stably labeled,and can be used for ideal tags for protein quantification.The interference of ion suppressive effect can be corrected by standard curve,and this suppressive effect has no influence on protein quantification before the ratio of two metal ions reached to 6.The improved strategy provides a technology that can be used in protein quantification not only at the peptide level,but also at the protein level.