Easy nlc 1000 liquid

Phage virion proteins were extracted from phages purified with polyethylene glycol (10¹⁰ pfu/mL) using chloroform-water-methanol extraction (1:1:0.75, v/v/v). The resulting protein pellet was resuspended in 20 µL of 10 mM Tris-HCl pH 6.8 and trypsinized using a gel-free method. For this, 10 µL of protein sample was mixed with 25 µL denaturation buffer (50 mM Tris-HCl pH 8.5, 6 M urea, 8.5 mM DTT) and incubated for 1 h at 56 °C in a water bath. After adding 25 µL 100 mM iodoacetamide (Sigma Aldrich, St. Louis, MO, USA) in 50 mM NH4HCO3 and 150 µL 50 mM NH4HCO3, the samples were incubated for 45 min in the dark, followed by adding 0.8 µg trypsin (Promega, Madison, WI, USA) to the samples and incubating overnight at 37 °C. Mass spectrometry analysis was performed on an Easy-nLC 1000 liquid chromatograph, coupled to a mass calibrated LTQ-Orbitrap Velos Pro via a Nanospray Flex ion source (all Thermo Fisher Scientific) using sleeved 30 µm ID stainless steel emitters. Peptides were identified with SEQUEST v1.4 (Thermo Fisher Scientific) and Mascot v2.5 (Matrix Sciences) and a database containing all possible translated open reading frames (ORFs), as identified using ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), and compared with the GenBank nr (non-redundant) protein database using the BLASTP.