Proteom discoverer 1

Proteins were mixed, alkylated by acrylamide and further processed as described [31 (link)]. Peptide samples were analysed with a shot-gun approach and data dependent analysis in a LC-MS system (RSLC, LTQ Orbitrap Velos, both Thermo Fisher) as recently described [31 (link)]. Raw MS data were processed using Proteom discoverer 1.4 (Thermos Scientific) and Max Quant software (version 1.5) [32 (link)] and a data base containing human and viral proteins and common contaminants. Proteins were stated identified by a false discovery rate of 0.01 on protein and peptide level.

Whole cell lysates from Kasumi-1/ctrl and Kasumi-1/shRE were prepared in RIPA buffer (50mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS), supplemented with complete Protease Inhibitor Cocktail (Roche), on day twelve after lentiviral transduction. Protein concentration was determined by DC-Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions and 50 μg protein per sample were boiled for five minutes in 2x NuPage LDS sample buffer (Invitrogen). Proteins were alkylated by acrylamide and further processed as described [32 (link)]. Peptide samples were analyzed with data dependent analysis in an LC-MS system (RSLC, LTQ Orbitrap Velos, both Thermo Fisher) as described recently [32 (link)]. Raw MS data were processed using Proteom discoverer 1.4 (Thermo Scientific) and Max Quant software (version 1.5, Cox and Mann 2008) and reviewed human and viral entries of the SwissProtUniprot database containing common contaminants. Proteins were stated identified by a false discovery rate of 0.01 on protein and peptide level.

For LC–MS analysis, >1 μg His-tagged SgG2 was used, purified by affinity chromatography from the supernatant of Hi5 insect cells, as described before [8 (link)]. Prior to analysis, SgG2 was separated by SDS–PAGE. Protein bands were cut out and in-gel digested with trypsin, AspN or successively with both enzymes as described elsewhere [18 (link)]. Extracted peptides were analysed with data-dependent analysis in an LC–MS system (nanoRSLC, LTQ Orbitrap Velos, both Thermo Fisher Scientific). Raw MS data files were processed using Proteom discoverer 1.4 (ThermoFisher Scientific Inc., Waltham, MA, USA) software (version 2.2,) and a data base containing insect entries and complete amino acid sequence of SgG2. Peptides were identified by a false discovery rate of <0.01 and MS2 spectra were checked visually.