Invitrosol

HEK293 were treated for 6 h with 10 μM MG132 to allow the accumulation of FBXW7 interacting proteins. Cells were then lysed in lysis buffer (50 mM Tris-HCl, ph 7.5, 150 mM NaCl and 2 mM EDTA) containing invitrosol (Invitrogen), PMSF, NaF, NaVO3 and proteinase inhibitors (Sigma). Tandem purification was performed. First immunoprecipitation was carried out overnight with anti-FLAG agarose beads. Next day, beads were washed and FLAG-HA-FBXW7 was eluted with 3xFLAG peptide. The eluate was used for a second immunoprecipitation with anti-HA agarose beads. Beads were thoroughly washed and subsequently digested for MS analysis.

HEK lysate was submitted to protein based fractionation by addition of organic solvent into ten protein fractions, effectively reducing the sample complexity. Protein pellets were washed with acetone and digested with trypsin. Dried pellets were dissolved in 8 M urea/100 mM Tris, pH 8.5. Proteins were reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Sigma-Aldrich) and alkylated with 10 mM iodoacetamide (Sigma-Aldrich). Proteins were digested overnight at 37 °C in 2 M urea/100 mM Tris, pH 8.5, 1 mM CaCl₂ with trypsin (Promega) in a ratio of 1:100 (enzyme:protein). Digestion was stopped with formic acid, 5 % final concentration. Debris was removed by centrifugation.
For the saliva and the rat brain samples, about 200 micrograms of proteins were solublized with 8 M urea/Invitrosol (Invitrogen, Calsbad, CA), reduced with 10 mM dithiothreitol, alkylated with 10 mM iodoacetomide, diluted with 4 volumes of 100 mM Tris-HCl, and then digested with trypsin overnight. After digestion, the pH was adjusted to ~ 2.5 using 90% formic acid. Sixty micrograms of protein digest from each sample was analyzed by MudPIT.