Mascot search engine program

LC–MS/MS data were searched against an in-house protein sequence database containing Swiss-Prot and the protein constructs specific to the experiment, using the Mascot search engine program (Matrix Science, version 2.4). Database search parameters were set with a precursor tolerance of 5 p.p.m. and a fragment ion mass tolerance of 0.8 Da. Variable modifications for oxidized methionine, carbamidomethyl cysteine, pyroglutamic acid, and deamination of glutamine/asparagine were included. MS/MS data were validated using the Scaffold program (version 5, Proteome Software Inc.).

Mass spectrometry was performed by the LMB Mass Spectrometry Facility. Briefly, peptides from in situ trypsin digestion were extracted in 2% formic acid/2% acetonitrile mix. Digests were analyzed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC and C18 Acclaim PepMap100 nanoViper (Thermo Scientific Dionex). LC-MS/MS data were searched against a protein database (UniProt KB) with the Mascot search engine program (Matrix Science). MS/MS data were validated using the Scaffold program (Proteome Software). MALDI-TOF mass spectrometric measurements were carried out in positive ion mode on an Ultraflex III TOF-TOF instrument (Bruker Daltonik), using sinapinic acid (Sigma) as matrix.

For affinity purification of UBR5-associated proteins, 20x 175 cm² flasks of HEK293T cells transfected with UBR5 or control baits were used for each experiment. Cells were lysed in 40 mL lysis buffer (20 mM Tris-HCl pH 7.4, 10% glycerol, 100 mM NaCl, 5 mM NaF, 2 mM Na₃PO₄, 0.2% Triton X-100, protease inhibitor cocktail), and sonicated 10x 10 s at 40% intensity with a Branson 250 Sonifier. Cell lysates were clarified by centrifugation (21,000x g, 30 min, 4°C) and incubated (while rotating) for 2 hr with Flag affinity gel at 4°C. Immunoprecipitates were washed 5x with lysis buffer, and subsequently eluted with lysis buffer supplemented with 250 μg mL⁻¹ 3xFlag-Peptide. Eluates were boiled in LDS sample buffer and resolved on 4%–12% Bis-Tris SDS-polyacrylamide gels. These were stained with Imperial Protein Stain, and gel lanes cut into 1-2 mm slices for in situ trypsin digestion. Resulting peptides were extracted in 2% formic acid / 2% acetonitrile mix. Digests were analyzed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC and C18 Acclaim PepMap100 nanoViper (Thermo Scientific Dionex). LC-MS/MS data were searched against a protein database (UniProt KB) with the Mascot search engine program (Matrix Science). MS/MS data were validated using the Scaffold program (Proteome Software).