Mass spectrometry analysis of phage-infected cells.

Proteins were separated by electrophoresis in Criterion XT MOPS 12% SDS-PAGE reducing gels (Bio-Rad), with subsequent protein visualization by staining with Coomassie blue. Each gel lane was divided into six slices. After destaining, proteins in the gel slices were reduced with TCEP [Tris(2-carboxyethyl)phosphine hydrochloride] and then alkylated with iodoacetamide before digestion with trypsin (Promega). High-pressure liquid chromatography/electrospray ionization/tandem mass spectrometry was accomplished on a Thermo Fisher LTQ Orbitrap Velos Pro mass spectrometer or a Thermo Fisher Orbitrap Fusion Lumos Tribrid mass spectrometer. Mascot (Matrix Science, London, UK) was used to search the mass spectrometry (MS) files against a locally generated SPN3US protein database that had been concatenated with the Swiss-Prot database (2012_11_170320; version 51.6).

Subset searching of the Mascot data, determination of probabilities of peptide assignments, and protein identifications were accomplished by Scaffold (Proteome Software). MS data files for each entire gel lane were combined via the “MudPIT” option. The results for identified SPN3US proteins, numbers of unique peptides, total spectra, and sequence coverage for each experiment were exported from Scaffold with the following quality filters: peptide, 95%; protein, 99.9%; and minimum number of peptides, 2 (or 1 peptide in selected instances). An estimate of the relative amount of each SPN3US protein produced in each sampling period was calculated by dividing the total number of spectra identified at each time point by protein molecular weight (SC/M) as performed previously for phages 0305ϕ8-36 (45), 201ϕ2-1 (36, 45), RIO-1 (46), and ϕKZ (11). These data were also normalized for the highest number of spectra identified for each protein, and these data were displayed using GenVision version 14.1.0.118 (DNASTAR, Inc., Madison, WI). The results for identified Salmonella proteins for each experiment were exported from Scaffold (1% false discovery rate).

The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE (47) partner repository with the data set identifiers PXD011697 (time series of SPN3US-infected and control cultures of S. Typhimurium analyzed on the Lumos mass spectrometer), PXD011521 (SPN3US-infected and control cultures of S. Typhimurium at Time-20 analyzed on the Orbitrap mass spectrometer), and PXD011520 (ϕKZ-infected and control cultures of P. aeruginosa at Time-20 analyzed on the Orbitrap mass spectrometer).