Caspases,Paracaspases, and Metacaspases Methods and Protocols

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4 Notes

1. When preparing cell lysates, add protease and specifi c caspase
   inhibitors to eliminate any residual proteolytic activity. When
   possible prepare the samples and run the gel in the same day.


2. If enough lanes are available, run the sample in duplicate so
   you will have a back-up if something goes wrong during the
   mass spectrometric analysis.


3. When handling the gel and performing the in-gel digestions,
   make sure to always wear gloves and minimize exposure of the
   gel bands to possible keratin contamination.


4. When cutting the gel, make sure to keep the gel hydrated
   enough to prevent curling/shrinking, if you notice this hap-
   pening splash a little high-purity water onto the gel.


5. Make sure all glassware is clean and free of any detergents, which
   could negatively impact the mass spectrometric analysis.

References

1. Dix MM, Simon GM, Cravatt BF (2008) Global
   mapping of the topography and magnitude of
   proteolytic events in apoptosis. Cell
   134:679–691


2. Simon GM, Dix MM, Cravatt BF (2009)
   Comparative assessment of large-scale pro-

teomic studies of apoptotic proteolysis. ACS

Chem Biol 4:401–408

3. Cociorva D, Tabb DL, Yates JR (2006)
   Validation of tandem mass spectrometry data-
   base search results using DTASelect. Curr
   Protoc Bioinformatics 13.4.1–13.4.14, supple-
   ment 16, Johnwiley and Sons

Melissa M. Dix et al.

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