Sample Processing Procedure 1. Cells grown in Middlebrook7H9 medium until stationary phase(OD600=1.9). 2. Pellet cells, pour off supernatant, wash with PBS, pellet again. 3. Resuspend in LYSIS buffer (between 1-2ml/gram cells). LYSIS buffer: 25 mM Tris HCL pH 7.5 2.5 mM DTT 1.0 mM EDTA, disodium 0.02%(w/v) Brij 35 1X CPICSI (Calbiochem Protease cocktail set I) 4. break cells with .1mm glass beads. 5. Centrifuge 20,000g, 30 minutes (at 25degC). 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 90deg C for 5 min, cool to 37 degC. 8. Add trypsin solt'n (Sigma T-6567). 1:100 w/w of trypsin to protein. Digest for ~18hrs, 37degC. 9. Centrifuge in eppendorf, 5 min @ 10,000g and take supernatant. 10. Dilute (1/4, 1/10, or 1/20) into Mass Spec buffer C and analyze. 11. Store samples at -80degC when not running.