1. Considerations for Sample Preparation

2. Protein Extraction Mechanical grinding Detergents Other buffers Sonication

3. http://www.piercenet.com/browse.cfm?fldID=FA97 D803-6953-48E4-A7BD-6947D35FE83B http://www.piercenet.com/browse.cfm?fldID=FA97 D803-6953-48E4-A7BD-6947D35FE83B

4. Considerations for mass spectrometry Salts and buffers in high concentrations can cause ion suppression and adduct formation in electrospray mass spectrometry Detergents can interfere with reversed phase separations Proteases

5. Inhibitors Most cells have endogenous proteases that can indiscriminately cleave proteins once cellular structure is disrupted The same applies to many PTMs (phosphatases, deacetylases, etc)

6. Salts

7. Medicago with detergent

8. MS 2 of detergent

9. Lysis buffer Compatible lysis buffer 8 M Urea, 50 mM Tris pH 8.5, 5 mM CaCl 2, 50-100 mM NaCl, plus inhibitors

10. Detergents Mass Spec compatible: RapiGest (Waters) ProteaseMax (Promega) For other detergents, you need a clean up step prior to LC-MS FASP Precipitation (acetone, chloroform/methanol)

12. Desalting C18 reversed phase material