Proteomic Profiling of HSV-1 Infection in DRG Neurons

A total of 80,000 cultured DRG neurons per treatment (40,000 neurons/well and two wells were combined for each experiment) were infected with HSV-1 KOS or HSV-1 n212, or were uninfected, and treated with MG132 (Cbz-Leu-Leu-Leucinal) to inhibit the ubiquitin-proteasomal degradation complex and preserve ubiquitinated proteins post-inoculation. The infection progressed for 8 h prior to protein harvesting in 125 μL non-denaturing lysis buffer (20 mM Tris HCl pH8, 1% NP-40, 2 mM EDTA) with MG132, PR619 (2,6-Diaminopyridine-3,5-bis(thiocyanate)) broad-spectrum deubiquitinating enzyme inhibitor to prevent the removal of ubiquitin moieties after collection, and Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Samples were incubated for 12 h with FK2 anti-ubiquitin antibody covalently conjugated to Invitrogen Dynabeads (Thermo Fisher Scientific), according to the manufacturer’s protocol. Immunoprecipitated samples were rinsed 3 times with non-denaturing buffer and resuspended in non-denaturing lysis buffer with MG132, PR619, and Halt Protease & Phosphatase Inhibitor. Samples were shipped overnight to MSBioworks (Ann Arbor, MI, USA) for LC-MS/MS analysis. Each sample was eluted in 70 μL 1.5× NuPage LDS Sample Buffer (Thermo Fisher Scientific) and boiled at 100 °C for 15 min, followed by clarification via centrifugation. Half of each sample was processed by SDS-PAGE using a 10% Bis-Tris NuPage Mini-gel (Thermo Fisher Scientific) with an MES buffer system. A 2 cm gel space was excised into ten bands, washed with 25 mM ammonium bicarbonate and acetonitrile, reduced with 10 mM dithiothreitol at 60 °C, alkylated with 50 mM iodoacetamide at room temperature, digested with trypsin at 37 °C for 4 h, quenched with formic acid, and finally analyzed using a nano LC-MS/MS with Waters M-Class LC system interfaced to a Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Each sample was analyzed for 5 h. The infection was repeated a second time, using neuronal cultures performed on a different day and following the identical processes and protocols to generate a replicate set of mass spectrometry data.