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Herpesviral capsids are assembled in the host cell nucleus before being translocated to the cytoplasm where they undergo further maturation. The crossing of nuclear envelope represents a major event that requires formation of the nuclear egress complex (NEC). We investigated the protein composition of specific HCMV-NEC complex in infected infected human primary fibroblasts.
Sample Processing Protocol
Immunoprecipitation experiments of the NEC were realized and the protein composition determined using proteomic analyses. Proteins were stacked in the top of a SDS-PAGE gel before in-gel digestion. Resulting peptides were analysed by online nanoLC-MS/MS (Ultimate 3000, Dionex and LTQ-Orbitrap Velos pro, Thermo Fischer Scientific).
Data Processing Protocol
Peptides and proteins were identified using Mascot (v. 2.4) and filtered using IRMa (v. 1.31.1): conservation of rank 1 peptides, peptide identification FDR < 1% (as calculated on peptide scores by employing the reverse database strategy), and minimum of 1 specific peptide per identified protein group. Filtered results were uploaded into a relational mass spectrometry identification database (MSIdb) before compilation, grouping and comparison of the protein groups from the different samples using an homemade tool (hEIDI v. 1.14.3).
Milbradt J, Kraut A, Hutterer C, Sonntag E, Schmeiser C, Ferro M, Wagner S, Lenac T, Claus C, Pinkert S, Hamilton ST, Rawlinson WD, Sticht H, Coute Y, Marschall M. Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus. Mol Cell Proteomics. 2014 Jun 26. pii: mcp.M113.035782 PubMed: 24969177