Genomics and Proteomics of Mycobacteriophage Patience, an Accidental Tourist in the Mycobacterium Neighborhood

Pope, Welkin H.; Jacobs-Sera, Deborah; Russell, Daniel A.; Rubin, Daniel H. F.; Kajee, Afsana; Msibi, Zama N. P.; Larsen, Michelle H.; Jacobs, William R.; Lawrence, Jeffrey G.; Hendrix, Roger W.; Hatfull, Graham F.

HPLC-MS/MS. Five milliliters of exponentially growing M. smegmatis mc 2 155 (optical density at 600 nm [OD 600 ] of 0.4) in 7H9-ADC medium (30) was concentrated to a 500-l volume via low-speed centrifugation and infected with Patience at a multiplicity of infection (MOI) of 100. Phage particles were allowed to adsorb for 15 min, and then 4.5 ml of fresh 7H9 medium was added to the culture and incubated with shaking for 3 h at 37°C; the OD 600 was monitored throughout to follow cell growth and lysis. At 30 min and 150 min postadsorption, a 1-ml aliquot was removed from the culture, the cells were pelleted via centrifugation (1 min, 14,000 rpm in a microcentrifuge), and the supernatant was removed. The cell pellet was frozen at Ϫ80°C and then shipped overnight on wet ice to the University of California, Davis Proteomics Core (UCDPC) (http://proteomics.ucdavis.edu). There, the cells were lysed via a MagNA Lyser, the insoluble fraction was removed, and the soluble proteins were precipitated, digested with trypsin, and cleaned up using a MacroSpin column. The peptides were then separated using an Easy-LC II high-pressure liquid chromatography (HPLC) system and loaded into a Q Exactive Orbitrap mass spectrometer with a Proxeon nanospray source (Thermo) for tandem MS analysis. Detected spectra and fragmentation profiles were matched against a database comprised of a six-frame translation of the Patience genome, the annotated proteins of M. smegmatis mc 2 155, and UniProt using X! Tandem. Peptide matches were analyzed using Scaf-fold4. The "Relaxed" settings (as reported in Table S1 in the supplemental material) used a peptide false discovery rate (FDR) of 1% and a protein FDR of 5%; the "Stringent" settings used a peptide FDR of 0.1% and a protein FDR of 0.6%. Estimation of relative protein abundance was determined by normalizing the total number of spectra detected to the gene size.