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

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.