Validation of Mycobacteriophage Genome Annotation by Mass Spectrometry

Abstract

Mycobacteriophages are viruses that infect Mycobacteria. Phage proteins are traditionally studied by nucleotide sequence based methods. Phage host interactions are complex and thus it is important to validate in silico annotations with a wet lab also examining the phage-host interaction. In this research, fifteen mycobacteriophages isolated through the Howard Hughes Medical Institute (HHMI) Science Education Alliance (SEA) were examined by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Phage samples were incubated with host cell culture overnight, proteins extracted, digested and subjected to LC-MS/MS. The results were analyzed using Mascot and peptides of host cells and phages were identified by searching MS spectral data in separate bioinformatics databases. We found the proteomic data could be used to cluster new phages based upon protein comparison with previously characterized phage genomes. We detected unexpected peptides that were not predicted by the genome annotation. Our findings highlight the importance to examining the phage-host system and future research will explore the impact of phage-host interaction and the phage life cycle on the expression of phage proteins. This program has received funding from HHMI SEA, Purdue University COT and ABE.