Biology Reference
In-Depth Information
4. Add 40 μl alkylating solution. Incubate for 15 min in the dark
at room temperature. Take off and discard the supernatant.
5. Add 100 μl destaining solution. Shake for 10 min at room
temperature. Take off and discard the supernatant.
1. Carefully dice gel slices into smaller pieces (approx. 2-3 mm
2
in size).
2. Add 50 μl destaining solution. Shake for 10 min at room tem-
perature. Take off and discard the supernatant.
3. Dry gel pieces in a vacuum concentrator.
4. Add 30-100 μl trypsin solution to a protein-to-trypsin ratio of 20
to 1 (the gel pieces should be covered with the trypsin solution).
Incubate overnight at room temperature (
see
Note 10
).
3.4.3
Tryptic Digestion
1. Add extraction solution (same volume as trypsin solution).
Shake for 40 min at room temperature. Take off the superna-
tant and transfer to a fresh 1.5 ml Eppendorf tube (do not
discard!).
2. Add extraction solution (same volume as trypsin solution).
Shake for 10 min at room temperature. Take off the superna-
tant and combine it with the first supernatant (
step 1
) (do not
discard!).
3. Dry the peptides (collected supernatants) in the vacuum
concentrator.
3.4.4 Extraction
of Peptides
1. Dissolve dried peptides in 20 μl 2 % ACN and 0.1 % FA.
2. Incubate for 5 min in an ultrasonic bath.
3. Centrifuge for 3 min at 17,000 ×
g
and transfer supernatant
into an HPLC vial suitable for the autosampler.
4. For the liquid chromatography (LC) inject 1-8 μl of the
sample.
5. Loading: 5 min at 6 μl/min on the trap column.
6. Gradient (this is a suggestion for a complex protein mixture):
Use a column flow rate of 0.240 μl/min (0-5 min 8 % solvent
B; 5-90 min 8-40 % solvent B; 90-95 min 40-85 % solvent B;
95-105 min 85 % solvent B; 105-110 min 85-8 % solvent B;
110-120 min 8 % solvent B).
7. The acquisition: Cycle of survey scan followed by four data-
dependent scans of the four most abundant peaks. MS data is
acquired over the complete LC run.
8. Cycle time: 30 ms, scan range: 500-2,000 Da.
9. CID; normalized collision energy: 35.0 V.
3.5
MS Analysis
