Jalili Pegah R, Ball Haydn L
Protein Chemistry Technology Center, University of Texas, Southwestern Medical Center, Dallas, Texas 75390-8816, USA.
J Am Soc Mass Spectrom. 2008 May;19(5):741-50. doi: 10.1016/j.jasms.2008.02.004. Epub 2008 Feb 26.
To improve the detection of phosphorylated peptides/proteins, we developed a novel protocol that involves the chemical derivatization of phosphate groups with a chemically engineered biotinylated-tag (biotin-tag), possessing three functional domains; a biotin group for binding to avidin, a base-labile 4-carboxy fluorenyl methoxycarbonyl (4-carboxy Fmoc) group, and a nucleophilic sulfhydryl moiety on the side-chain of cysteine. Using this approach, the derivatized, enzymatically digested peptides were selectively separated from unrelated sequences and impurities on immobilized avidin. Unlike previously published phosphopeptide enrichment procedures, this approach upon treatment with mild base liberates a covalently bound Gly-Cys analog of the peptide(s) of interest, exhibiting improved RP-HPLC retention and MS ionization properties compared with the precursor phosphopeptide sequence. The results obtained for a model peptide Akt-1 and two protein digests, demonstrated that the method is highly specific and allows selective enrichment of phosphorylated peptides at low concentrations of fmol/microL.
为了提高磷酸化肽段/蛋白质的检测效率，我们开发了一种新方法，该方法涉及使用一种化学工程化的生物素化标签（生物素标签）对磷酸基团进行化学衍生，这种标签具有三个功能域：一个用于与抗生物素蛋白结合的生物素基团、一个对碱不稳定的4-羧基芴甲氧羰基（4-羧基芴甲氧羰基）基团以及一个位于半胱氨酸侧链上的亲核巯基部分。使用这种方法，衍生化的、经酶消化的肽段能够从固定化抗生物素蛋白上的无关序列和杂质中选择性分离出来。与先前发表的磷酸肽富集方法不同，该方法在用弱碱处理后会释放出目标肽段的共价结合的甘氨酸-半胱氨酸类似物，与前体磷酸肽序列相比，其在反相高效液相色谱（RP-HPLC）中的保留和质谱电离特性得到了改善。对模型肽Akt-1和两种蛋白质消化产物的检测结果表明，该方法具有高度特异性，能够在低至飞摩尔/微升的浓度下选择性富集磷酸化肽段。
