Briefly, proteins were reduced at 4 C for 1 h with 10 mM DTT and alkylated nevertheless by incubation with 25 mMiodoacetamide at 4 C for 1 h in the dark. Proteins were recovered through acet one precipitation and digested with trypsin at an enzyme substrate ratio of 1 50 in 50 mM NH4HCO3 overnight at 37 C. Tryptic peptides were analysed by bidimensional chromatography and online MS MS, as described elsewhere, except that only 3 suc cessive salt plugs of 25, 100 and 800 mM NH4Cl were used. Peptides were analyzed using the peptide scan op tion of an HCT ultra ion Trap, consisting of a full scan mass spectrometry and MS MS scan spectrum acquisitions in ultrascan mode. Peptide fragment mass spectra were acquired in data dependent AutoMS mode with a scan range of 100 2,800 mz, three averages, and 5 precursor ions se lected from the MS scan 300 1500 mz.
Precursors were actively excluded within a 0. 5 min window, and all singly charged Inhibitors,Modulators,Libraries ions were excluded. Inhibitors,Modulators,Libraries Peptide peaks were detected and deconvoluted Inhibitors,Modulators,Libraries automatically using Data Analysis 2. 4 software. Mass lists in the form of Mascot Gen eric Files were created automatically and used as the input for Mascot MSMS Ions searches of the NCBInr database Inhibitors,Modulators,Libraries release 20120809 using an in house Mascot 2. 2 server. The default search parameters used were Taxonomy Bony vertebrates or Cyprinivirus. En zyme Trypsin. Maximum missed cleavages 1. Fixed modifications Carbamidomethyl . Variable modifica tions Oxidation . Peptide tolerance 1. 2 Dalton . MSMS tolerance 0. 6 Da. Peptide charge 2 and 3 . Instrument ESI TRAP.
All data were also searched against the NCBI Inhibitors,Modulators,Libraries bony vertebrate database in order to de tect host proteins. Only proteins identified with p value lower than 0. 05 were considered, and single peptide iden tifications were systematically evaluated manually. In addition, the emPAI was calculated to estimate pro tein relative abundance in the culture supernatant. Production of CyHV 3 ORF134 recombinants CyHV 3 recombinants were produced using prokaryotic recombination technologies. The FL BAC plas mid was used as parental plasmid. In this plasmid, the BAC cassette is inserted in ORF55 encoding thymidine kinase. ORF134 recombinant plasmids were pro duced using two steps galactokinase gene positive negative selection in bacteria as described previously. The first recombination process consisted to replace ORF134 by galK resulting in the FL BAC ORF134 Del galK plasmid.
Recombination was achieved using the H1 galK H2 recombination cassette which consisted of the galK gene flanked by 50 bp sequences homologous to CyHV 3 genome regions flanking ORF134 deletion. H1 galK H2 recom bination cassette was produced by PCR using the pgalK vector as template. Primer 134 galK F consisted of nucleotides 229836 229885 of CyHV 3 genome and 1 24 of selleck inhibitor the pgalK vector. Primer 134 galK R consisted of nucleotides 229262 229311 of the CyHV 3 genome and nucle otides 1212 1231 of the pgalK vector.