Mass Spectrometry of Phage Particles

For mass spectrometry, 5 μL of concentrated phage lysate (6 × 10¹⁰ PFU/mL; protein concentration determined as 2.5 mg/mL using a Pierce BCA kit) and purified phage particles (2 × 10¹² PFU/mL; protein concentration of 2.8 mg/mL) were used for trypsin digestion. The samples were prepared for mass spectrometry essentially as described in [45 (link)]. Phage samples were mixed with 8 M urea–100 mM ammonium bicarbonate to a final volume of 50 μL, and the cysteine bonds were reduced with 5 mM tris(2-carboxyethyl)phosphine (TCEP) (37 °C for 60 min) with subsequent alkylation using 10 mM iodoacetamide (22 °C for 30 min). Ammonium bicarbonate, at 100 mM, was used to dilute the urea concentration of the samples to 1.5 M. Proteins were digested for 18 h at 37 °C with sequencing grade trypsin (Promega). Formic acid (10%) was used to lower the pH of the samples to 3.0, and the peptides were subsequently purified with C18 reverse-phase spin columns according to the manufacturer’s instructions (Microspin Columns, Harvard Apparatus). The dried peptides were reconstituted in 2% acetonitrile and 0.2% formic acid prior to mass spectrometric analyses.