Identification of an enterovirus recombinant with a torovirus-like gene insertion during a diarrhea outbreak in fattening pigs

Conceição-Neto, Nádia; Theuns, Sebastiaan; Cui, Tingting; Zeller, Mark; Yinda, Claude Kwe; Christiaens, Isaura; Heylen, Elisabeth; Van Ranst, Marc; Carpentier, Sebastien; Nauwynck, Hans J.; Matthijnssens, Jelle

Peptides were separated on an Ultimate 3000 UPLC system (Dionex, ThermoScientific) equipped with an Acclaim PepMap100 pre column (C18 3 lm-100 Å , Thermo Scientific) and an C18 PepMap RSLC (2 lm, 50 lm_15 cm, ThermoScientific) using a linear gradient (300 ll/min) of 0-4% buffer B (80% ACN, 0.08% FA) in 3 min, 4-10% in 12 min, 10-35% in 20 min, 35-65% in 5 min, 65-95% in 1 min 95% for 10 min, 95-5% in 1 min, 5% 10 min. Then the separated peptides were analyzed in the Orbitrap QE operated in positive ion mode (nanospray voltage 1.5 kV, source temperature 250 C). The instrument was first operated in data-dependent acquisition (DDA) mode on the pool of synthetic peptides ordered (PEPotec SRM Custom Peptide Libraries, Thermo Scientific) and an inclusion list based on the m/z and RT of the synthetic peptides detected was created (Supplementary Tables S1 and S2 ). For DDA, MS scans were performed at a resolution of 70,000 fwhm for the mass range of m/z 400-1,600 for precursor ions, followed by MS/ MS scans of the top 10 most intense peaks with þ2, þ 3, and þ4 charged ions above a threshold ion count of 16,000 at 35,000 of resolution. MS/MS was performed using normalized collision energy (NCE) of 25% with an isolation window of 3.0 m/z, an apex trigger 5-15 s, and a dynamic exclusion of 10 s. Data were acquired with Xcalibur 2.2 software (Thermo Scientific). Then the sample and synthetic peptides were re-ran in MRM/MSMS mode (Supplementary Table S2 ).