title: 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.
abstract: 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.
authors: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
authors: Ko-Yi
authors: KY
authors: Chien
authors: Hsiao-Ching
authors: HC
authors: Liu
authors: Michael B
authors: MB
authors: Goshe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins / Q000032:analysis / Q000378:metabolism; D002642:Chick Embryo; D002853:Chromatography, Liquid; D005347:Fibroblasts; D054884:Host-Pathogen Interactions / Q000502:physiology*; D057927:Hydrophobic and Hydrophilic Interactions; D025922:Mardivirus; D008380:Marek Disease / Q000276:immunology / Q000378:metabolism / Q000821:virology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D055672:Static Electricity; D053719:Tandem Mass Spectrometry; D014764:Viral Proteins / Q000032:analysis / Q000378:metabolism
chemical_list: D030161:Avian Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D014764:Viral Proteins