A host independent strain of B. bacteriovorus (ATCC 25631) obtained from American Type Culture Collection was grown on culture medium containing peptone (1%) and yeast extract (0.3%). All incubations were at 30˚^. Liquid cultures were grown on a shaker. The MAC-1 phage titer was determined according to Adams [15] and high titer phage was prepared according to Hershey et al. [16] except that large petri plates (150 × 15 mm) were used. The original MAC-1 bacteriophage was provided by Dr. Mary Althauser [10].

Restriction enzymes, T4 DNA ligase and other nucleic acid enzymes used for DNA manipulation were from US Biological or New England Biolabs. DNA sequencing kits based either on Sequenase or Themo Sequenase were from Amersham/Pharmacia Biochemical. Sources other materials have been described [12] [17]-[20].

Bacteriophage from high titer broth culture was precipitated with 2.5% NaCl and 10% polyethylene glycol. The precipitate was collected and dissolved in buffered saline [20 mM potassium phosphate (pH 6.8), 150 mM NaCl and 5 mM Mg acetate]. It was further purified by two consecutive cycles of isopycnic density gradient centrifugation in CsCl (beginning with density of 1.36 g/ml); the phage stock (with a titer of 6 × 10¹⁴ to 1 × 10¹⁵/ml) was used to isolate phage nucleic acid according to Maniatis et al. [18].

The sequence of phage genome was determined by using two step sequence system with [³⁵S] dATP using Sequenase or deltaTaq DNA polymerase [18] [21] [22]. In our latter sequencing work we used [³²P] labeled terminators and Thermo Sequenase [23] because in this system only properly terminated chains are labeled and visualized in an autoradiograph of a sequencing gel. The band intensities are remarkably uniform. Moreover, PCR based cycle sequencing requires very small amounts of the template DNA. There are very few, if any, artifacts generated in DNA sequence due to high secondary structures in DN template. Experimental details of the method have been described [23]. Final DNA sequence of phage genome was developed by sequencing both the strands.

Phage purified by two consecutive isopycnic density gradient centrifugations in CsCl was dialyzed against phosphate buffered saline to remove CsCl. Phage stock (5 × 10¹⁴ to 1 × 10¹⁵) was used to analyze phage proteins. For this purpose an aliquot of phage was treated with denaturing solution containing [0.1 mM Tris-HCl (pH 7.0); 5% β-Mercaptoethanol; 10% Sodium Dodecyl Sulfate and 10% glycerol] and sample was heated at 95˚C for 5 minutes to allow complete denaturation of phage protein and their dissociation from phage DNA. An aliquot of the sample was applied to SDS-polyacrylamide (11% - 12%) gel [24] and standard proteins were used as molecular weight markers in a separate lane. They were subjected to electrophoresis to resolve the proteins. Western blot of resolved proteins in SDS-polyacrylamide gel was performed as described before [17] except that [¹²⁵I] labeled protein A was used for protein detection by autoradiography.

Antibodies against the purified whole phage were prepared in rabbits using the standard immunization protocol [17] [18]. The antibodies against individual phage proteins resolved by SDS-gel electrophoresis were also raised. For this purpose individual stained protein band from SDS-gel were cut out, homogenized in tight fitting homogenizer and then injected in a rabbit. Final immunization results showed that both the neutralizing antibodies [15] and antibodies against individual proteins were elicited by immunization with the whole phage and individual proteins. Details of other procedures used in our studies have been described [17]-[21] [23].

Sample bands of interest from dried SDS-PAGE gel were scored and cut out with a razor blade and then were completely cut from the paper (See Figure 1 below). After rehydration in LC-MS grade water, the gel fragments were then subjected to in-gel trypsin digestion for LC-MS/MS as previously described [25]. Briefly, the gel pieces were washed with 200 μL of LC-MS Grade Water (Optima LC-MS, Fisher Scientific) for 30 s and destained with 2 × 200 μL of 50% Acetonitrile (ACN; Optima LC-MS Grade)/50 mM Ammonium bicarbonate at 60˚C, with intermittent mixing. The pieces were dehydrated with 100% ACN and allowed to air dry. Proteins were reduced and alkylated, in-gel with 25 mM DTT in 50 mM Ammonium bicarbonate (60˚C for 20 min) and 55 mM IAA in 50 mM Ammonium bicarbonate at room temperature in the dark for 20 min. Gel pieces were then washed with Optima water and dried. The dried gel pieces were rehydrated in 20 μL 12 ng/μL MS-grade Trypsin (ThermoPierce, San Jose, CA) /0.01% ProteaseMAX surfactant/50 mM ammonium bicarbonate mixture for 10 min at room temperature, overlaid with 30 μL 0.01% ProteaseMAX surfactant/50 mM ammonium bicarbonate and incubated at 50˚C for 1 h. Extracted peptides were transferred and the digestion halted by addition of 10% trifluoro-acetic acid to a final concentration of 0.5%. Peptide extracts were dehydrated and stored at −20˚C prior to analysis by LC-MS/MS.

Peptides were purified and concentrated using an on-line enrichment column (Thermo Scientific 5 μm, 100 μm ID × 2 cm C18 column). Subsequent chromatographic separation was performed on a reverse phase nanospray column (Thermo Scientific EASYnano-LC, 3 μm, 75 μm ID × 100 mm C18 column) using a 30 minute linear gradient from 10% - 30% buffer B (100% ACN, 0.1% formic acid) at a flow rate of 400 nanoliters/min. Peptides were eluted directly into the mass spectrometer (Thermo Scientific Orbitrap Velos) and spectra were collected over a m/z range of 400 - 2000 Da using a dynamic exclusion limit of 2 MS/MS spectra of a given peptide mass for 30 s (exclusion duration of 90 s). The instrument was operated in Orbitrap-LTQ mode where precursor measurements were acquired in the orbitrap (60,000 resolution) and MS/MS spectra (top 20) were acquired in the LTQ ion trap with a normalized collision energy of 35 kV. Compound lists of the resulting spectra were generated using Xcalibur 2.2 software (Thermo Scientific) with a S/N threshold of 1.5 and 1 scan/group.

Tandem mass spectra were extracted, charge state deconvoluted and deisotoped by ProteoWizard MsConvert (version 3.0). All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.3.01). Mascot was set up to search the MAC-1 data generated in all possible open reading frames (ORFs) from the genome sequence and the Uniprot databases assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 20 PPM. Oxidation of methionine, carboxymethylation of cysteine, was specified in Mascot as variable modifications.

Search results from all samples were imported and combined using the probabilistic protein identification algorithms 1 implemented in the Scaffold software (version Scaffold_4.4.1.1, Proteome Software Inc., Portland, OR). Peptide identifications were accepted if they could be established at greater than 50.0% probability by the Peptide Prophet algorithm of Keller et al. [26] with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 90% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al. [26]-[29]). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.

Instrument functionality and stability was monitored using the MassQC software (Proteome Software). This software uses a set of quantitative metrics developed by the National Institute of Science and Technology (NIST) in collaboration with the National Cancer Institute’s Clinical Proteomic Technologies for Cancer (CPTC) that monitor technical variability in mass spectrometry-based proteomics instrumentation 5. Quality control samples containing a mixture of 6 trypsin digested bovine proteins were injected at least once every 24 hours throughout the analysis, and the data from this run was analyzed using the MassQC software. Values for all metrics were within normal limits throughout the duration of the experiment indicating instrument stability and data robustness.