Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis
Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis
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DOI：
10.1002/elps.200600718
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发表时间：
2007-07
期刊：
影响因子：
2.9
通讯作者：
Houjiang Zhou;Ruijun Tian;M. Ye;Songyun Xu;Shun Feng;C. Pan;Xiaogang Jiang;Xin Li;Hanfa Zou-
中科院分区：
文献类型：
--
作者：
Houjiang Zhou;Ruijun Tian;M. Ye;Songyun Xu;Shun Feng;C. Pan;Xiaogang Jiang;Xin Li;Hanfa Zou-
磷蛋白的大规模表征需要高度特异性的方法来纯化磷酸肽，因为磷蛋白的丰度低，磷酸化的亚化学计量。提出了一种利用纳米ZrO 2颗粒富集磷酸肽的方法。这种方法的高度特异性被证明是由模型磷蛋白的磷脂酶的磷酸肽的分离。ZrO 2纳米颗粒对磷酸肽的强亲和力使得能够从复杂的肽混合物中特异性富集磷酸肽，其中磷酸肽的丰度比非磷酸肽低两个数量级。观察到ZrO 2纳米颗粒富集磷酸化肽的上级选择性比传统的固定化金属亲和色谱。飞摩尔磷酸肽消化产物可以富集氧化锆纳米粒子，并可以很好地检测MALDI质谱分析。进一步应用ZrO 2纳米颗粒从小鼠肝脏裂解物的胰蛋白酶消化中选择性地分离磷酸肽，用于通过纳升LC MS/MS（nano-LC-MS/MS）和MS/MS/MS进行磷酸蛋白质组分析。通过使用一系列严格标准的手动验证，鉴定了总共248个定义磷酸化位点和140个磷酸化肽。
Large‐scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano‐LC‐MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.