Considerations for Sample Preparation

Protein Extraction Mechanical grinding Detergents Other buffers Sonication

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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

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)

Salts

Medicago with detergent

MS 2 of detergent

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

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)

Desalting C18 reversed phase material