65- Add enough 55 mM iodoacetamide (made fresh) to cover gel
band (approx 200 μL). - Place tubes in the dark for 30 min at room temperature.
- Remove supernatant and wash gel bands 2–3× for 15 min in
500 μL 50:50 acetonitrile:100 mM ammonium bicarbonate. - Remove supernatant and add 50 μL acetonitrile to completely
dry gel band (should turn opaque). - Remove acetonitrile and speed vac for ~5 min or until slices are
completely dry. - Resuspend a 20 μg bottle of trypsin in 200 μL of trypsin resus-
pension buffer. - Dilute 1:10 with 25 mM ammonium bicarbonate (10 ng/μL
fi nal concentration of trypsin). - Add 200 ng of trypsin (20 μL) to dehydrated gel slices (or if
sure of exact protein concentration, use a 50:1 substrate/
enzyme ratio). - Incubate gel slices in trypsin for 10 min to rehydrate.
- Add enough 25 mM ammonium bicarbonate to cover gel
bands and place at 37 °C overnight. - The next morning transfer supernatant into clean tube and add
enough 5 % formic acid to cover gel bands and incubate at
room temperature for 15 min. - Transfer supernatant to the tube with supernatant from the
o/n digestion. - Add enough acetonitrile to cover gel bands (approx 100–
200 μL) and incubate at room temperature for 15 min. Transfer
supernatant to tube with digested peptides. - Repeat acetonitrile elutions until gel bands have become com-
pletely opaque (2–3 times). - Dry peptide sample to a fi nal volume of <10 μL in speed vac.
- At this stage peptides may be stored at −80 °C ( see Note 5 ).
- Cut 30 cm of 100 μm ID fused silica capillary tubing and burn
off 2–3 cm of polyimide coating using a fl ame. - Remove the burnt coating with methanol and a Kimwipe.
Place the tubing in the laser puller with the newly exposed sil-
ica in the laser’s path and pull two 5 μm tips. - Create a methanol Aqua C18-reverse-phase slurry in an
Eppendorf tube, and pressure-load the tips from the previous
step with 11 cm of Aqua C18 reverse-phase slurry. - Equilibrate the tip on an Agilent 1100 series HPLC using a
gradient of 40 % buffer A; 60 % buffer B to 100 % buffer A; 0 %
buffer B over 30 min, followed by a 10 min wash with 100 %
3.3 Preparing
Columns for Mass
Spectrometric
Analysis
Global Identifi cation of Caspase Substrates