Development and Application of a Phosphoproteomic Method Using Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC), IMAC, and LC-MS/MS Analysis to Study Marek's Disease Virus Infection
Development and Application of a Phosphoproteomic Method Using Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC), IMAC, and LC-MS/MS Analysis to Study Marek's Disease Virus Infection
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DOI：
10.1021/pr2002403
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发表时间：
2011-09-01
影响因子：
4.4
通讯作者：
Goshe, Michael B.
中科院分区：
文献类型：
--
作者：
Chien, Ko-yi;Liu, Hsiao-Ching;Goshe, Michael B.
马立克氏病（MD）是由马立克氏病病毒（MDV）引起的禽类肿瘤疾病。病毒在裂解周期和潜伏周期之间转换的机制仍在研究中；然而，翻译后修饰，尤其是磷酸化，被认为发挥着重要作用。此前，我们课题组利用强阳离子交换色谱结合反相液相色谱串联质谱（LC-MS/MS）研究了MDV感染后整体蛋白质组表达的变化（Ramaroson, M. F.; Ruby, J.; Goshe, M. B.; 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，以研究磷酸化蛋白质组中发生的变化，使用静电排斥-亲水相互作用色谱 (ERLIC) 来分级肽。鸡胚成纤维细胞 (CEF) 消化并纳入后续的 IMAC 富集步骤，以选择性靶向磷酸化肽进行 LC-MS/MS 分析。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标。单独使用 LC MS/MS 分析时，没有检测到 CEF 磷酸肽，而使用 ERLIC 分级分离时，所有已识别肽中只有 1.2% 被磷酸化。然而，将 IMAC 富集与 ERLIC 分级分离相结合，已鉴定的磷酸肽的百分比增加了 50 倍。总体而言，总共鉴定出 581 种独特的磷酸肽（p < 0.05），MDV 感染的 CEF 样本中的磷酸肽含量几乎是模拟感染对照的两倍，其中 11% 是 MDV 蛋白所独有的。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。
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 rnicrotubule-associated proteins may play in MDV infection mechanisms.