Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
J Proteome Res. 2011 Sep 2;10(9):4041-53. doi: 10.1021/pr2002403. Epub 2011 Jul 25.
Marek's Disease (MD) is an avian neoplastic disease caused by Marek's Disease Virus (MDV). The mechanism of virus transition between the lytic and latent cycle is still being investigated; however, post-translational modifications, especially phosphorylation, have been thought to play an important role. Previously, our group has used strong cation exchange chromatography in conjunction with reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the changes in global proteomic expression upon MDV infection (Ramaroson , M. F.; Ruby, J.; Goshe, M. B.; Liu , H.-C. S. J. Proteome Res. 2008, 7, 4346-4358). Here, we extend our study by developing an effective separation and enrichment approach to investigate the changes occurring in the phosphoproteome using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) to fractionate peptides from chicken embryo fibroblast (CEF) digests and incorporating a subsequent IMAC enrichment step to selectively target phosphorylated peptides for LC-MS/MS analysis. To monitor the multidimensional separation between mock- and MDV-infected CEF samples, a casein phosphopeptide mixture was used as an internal standard. With LC-MS/MS analysis alone, no CEF phosphopeptides were detected, while with ERLIC fractionation only 1.2% of all identified peptides were phosphorylated. However, the incorporation of IMAC enrichment with ERLIC fractionation provided a 50-fold increase in the percentage of identified phosphopeptides. Overall, a total of 581 unique phosphopeptides were identified (p < 0.05) with those of the MDV-infected CEF sample containing nearly twice as many as the mock-infected control of which 11% were unique to MDV proteins. The changes in the phosphoproteome are discussed including the role that microtubule-associated proteins may play in MDV infection mechanisms.
马立克氏病（MD）是一种由马立克氏病病毒（MDV）引起的禽类肿瘤性疾病。病毒从裂解周期到潜伏周期的转变机制仍在研究中；然而，翻译后修饰，特别是磷酸化，被认为起着重要作用。此前，我们的小组使用强阳离子交换色谱法与反相液相色谱-串联质谱法（LC-MS/MS）相结合，研究了 MDV 感染后全局蛋白质组表达的变化（Ramaroson，MF；Ruby，J；Goshe，MB；Liu，H.-C.S. J. Proteome Res. 2008, 7, 4346-4358）。在这里，我们通过开发一种有效的分离和富集方法来扩展我们的研究，使用静电排斥-亲水相互作用色谱（ERLIC）来分离鸡胚成纤维细胞（CEF）消化物中的肽，并结合随后的 IMAC 富集步骤来选择性地针对磷酸化肽进行 LC-MS/MS 分析，从而研究磷酸蛋白质组的变化。为了监测模拟和 MDV 感染的 CEF 样品之间的多维分离，使用酪蛋白磷酸肽混合物作为内部标准。单独使用 LC-MS/MS 分析，未检测到 CEF 磷酸肽，而仅通过 ERLIC 分级，只有 1.2%的所有鉴定肽被磷酸化。然而，将 IMAC 富集与 ERLIC 分级相结合，可使鉴定的磷酸肽百分比增加 50 倍。总的来说，共鉴定到 581 个独特的磷酸肽（p<0.05），其中 MDV 感染的 CEF 样品中含有近两倍于模拟感染对照的磷酸肽，其中 11%是 MDV 蛋白特有的。讨论了磷酸蛋白质组的变化，包括微管相关蛋白在 MDV 感染机制中可能发挥的作用。
