Sample Processing Procedure 1. Cells grown in YMD minimal medium at 30°C until early log phase (OD600=0.6-0.8). 2. Pellet cells, pour off supernatant, wash with PBS, pellet again. 3. Resuspend in 2 volumes of LYSIS buffer. LYSIS buffer: 20 mM Tris HCL pH 7.5 100mM NaCl 1X CPICSI (Calbiochem Protease cocktail set I) 4. Break cells with 4 volumes of glass beads and vortex 10 times at 4°C. 5. Centrifuge 20,000g, 30 minutes 4°C. 6. Collect supernatant and dilute into DIGESTION buffer to make 4mg/ml protein. DIGESTION buffer: 50 mM Tris HCL, pH 8.0 1.0 M Urea 2.0 mM CaCl2.2H20 7. Heat to 95°C for 10 min, cool to 37°C . 8. Add 1:50 w/w of trypsin ((Sigma T-6567) to protein and digest for ~18hrs at 37°C. 9. Centrifuge 30 min @ 20,000g and take supernatant. 10. 1:4 dilute into Mass Spec buffer C and analyze. 11. Store samples at -80°C when not running.