Experimental annotation of channel catfish virus by probabilistic proteogenomic mapping

Experimental identification of expressed proteins by proteomics constitutes the most reliable approach to identify genomic location and structure of protein-coding genes and substantially complements computational genome annotation. Channel catfish herpesvirus (CCV) is a simple comparative model for understanding herpesvirus biology and the evolution of the Herpesviridae. The canonical CCV genome has 76 predicted ORF and only 12 of these have been confirmed experimentally. We describe a modification of a statistical method, which assigns significance measures, q-values, to peptide identifications based on 2-D LC ESI MS/MS, real-decoy database searches and SEQUEST XCorr and DeltaC(n) scores. We used this approach to identify CCV proteins expressed during its replication in cell culture, to determine protein composition of mature virions and, consequently, to refine the canonical CCV genome annotation. To complement trypsin, we used partial proteinase K digestion, which yielded greater proteome coverage. At FDR <5%, for peptide identifications, we identified 25/76 previously predicted ORF using trypsin and 31/76 using proteinase K. Furthermore, we identified 17 novel protein-coding regions (7 potential ATG-initiated ORF). Most of these novel ORF encode small proteins (<100 amino acids). Directed, strand-specific reverse transcription real-time PCR confirmed RNA expression from 6/7 novel ATG-initiated ORF investigated.