Dionex u 3000 rslcnano hplc system

The Nano-LC-ESI-MS/MS analysis was performed using Quadrupole-Orbitrap instrument equipped with Dionex U 3000 RSLCnano HPLC system and Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific, Rockford, IL, USA). Peptide samples were pooled, and peptide fractionation was performed using HPLC system. Peptide fractions were then reconstituted in solvent A (Water/Acetonitrile 98:2 v/v, 0.1 % Formic acid) and then injected into LC-nano ESI-MS/MS system. Samples were first trapped onan Acclaim PepMap 100 trap column (100 μm × 2 cm, nanoViper C18, 5 μm, 100 Å) and washed with 98% solvent A at a flow rate of 4 μL/min for 6 min, and then separated on a PepMap RSLC C18 column (75 μm × 15 cm, nanoViper C18, 3 μm, 100 Å) at a flow rate of 300 nL/min. The LC gradient was run at 2% to 8% solvent B over 10 min, then from 8% to 30% over 55 min, followed by 90% solvent B for 4 min, and finally 2% solvent B for 20 min. Xcaliber software ver. 4.4 was used to collect MS data. The Orbitrap analyzer scanned the precursor ions with a mass range of 350–1800 m/z with 60,000 resolutions at m/z 200. Mass data are acquired automatically using proteome discoverer 2.5 (Thermo Fisher Scientific, Rockford, IL, USA).

Mass spectrometric analysis of the structural proteins of phage pEp_SNUABM_08 was performed as described [23 (link)]. The phage proteins prepared by implementing five rounds of freeze–thaw cycles were separated using 12% SDS-PAGE. Subsequently, the proteins spanning the entire lane were in-gel digested with trypsin (100 ng/μL). The resultant proteins derived from pEp_SNUABM_08 were identified by using a nano high-resolution LC-MS/MS spectrometer Q Exactive Hybrid Quadrupole-Orbitrap (Thermo Scientific, MA, USA) equipped with the Dionex U 3000 RSLC nano HPLC system.