Native Purification of yeast Met30 in the absence and presence of Cadmium followed by DSSO crosslinking and Mass spec analysis

HBTHMet30 cells were cultured in YEP + 2% galactose + 4µM Biotin at 30˚C and treated with 100 µM CdCl2 for 30 minutes. Native whole cells lysates were prepared in lysis buffer (50mM Hepes pH7.5, 200mM NaCl, 10% glycerol, 40mM imidazole, 0.2% Triton1mM dithiothreitol, 0.1mM orthovanadate, 1mM phenylmethylsulfonyl floride [PMSF], and 1mg/ml each leupeptin and pepstatin) and bound to Ni-resin. Proteins were eluted in the presence of 250mM imidazole. For cross-linking analysis, eluted HBTHMet30 was first bound to Streptavidin beads and then on-bead cross-linked with 0.5 mM DSSO in PBS buffer for 1 h at 37 °C. Bead-bound proteins were reduced with TCEP and alkylated with iodocetamide, digested by trypsin and cleaned-up with C18 tips prior to LC MSn analysis.