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.