Caspases,Paracaspases, and Metacaspases Methods and Protocols

63

precise sites-of-cleavage) enables prediction of the functional

consequences of cleavage events.

Here we describe a detailed protocol for using PROTOMAP,

which should be applicable to cell or tissue systems from a wide

range of biological models and even primary human specimens.

2 Materials

1. Tris-Glycine SDS-PAGE buffer (National Diagnostics,
   EC-870).


2. Loading dye.


3. 10 % acrylamide gel.


4. Ammonium bicarbonate.


5. Phosphate-buffered saline.


6. Prestained protein molecular weight marker.


7. Razor.


8. Clear glass plate.


9. Square glass container.


10. Formic acid.


11. Sequencing-grade modifi ed trypsin (Promega).


12. Trypsin resuspension buffer (Promega).


13. Iodoacetamide.


14. Tris (2-carboxyethyl) phosphine.


15. High-purity water.


16. Optima-grade acetonitrile.


17. Ammonium bicarbonate.


18. Speed vacuum.


19. 1.5 mL Eppendorf tubes.


20. Incubator (37 °C).


21. MS buffer A: 95 % high-purity water, 5 % optima-grade aceto-
    nitrile, and 0.1 % formic acid.


22. MS buffer B: 20 % high-purity water, 80 % optima-grade ace-
    tonitrile, and 0.1 % formic acid.


23. 5 μm C18 reverse-phase resin (Phenomenex).


24. Model P-2000 CO 2 laser puller (Sutter Instrument).


25. Agilent 1100 series HPLC.


26. LTQ ion trap mass spectrometer (Thermo Electron).


27. High pressure bomb.


28. Fused silica capillary tubing (365 μm outer diameter, 100 μm
    inner diameter (ID); Agilent, cat. no. 160-2255-10).

2.1 SDS-PAGE

2.2 In-Gel Digestion

2.3 Mass
Spectrometric
Analysis

Global Identifi cation of Caspase Substrates