Biology Reference
In-Depth Information
13. Digest for 3 h at 37 °C. According to the enzyme effi cacy, the
time of digestion could be shortened to 1 h, especially for the
protease mixture. When the digestion is complete, the experi-
ment can be interrupted until the next day and the samples
stored overnight at 4 °C.
14. After digestion, remove the solution around gel pieces from
each tube into a new one to gain the peptides produced.
15. Cover the gel pieces with extraction buffer 1 and sonicate in a
water bath for 15 min.
16. For each tube, remove the solution and collect it with the fi rst
portion.
17. Cover the gel pieces with extraction buffer 2 and sonicate in a
water bath for 15 min.
18. Remove the solution and collect with the fi rst two portions.
19. Lyophilize the joined extracts under vacuum.
20. Resolve the content of the tubes in 20
L of 1 % FA and soni-
cate for 10 min to complete the dissolution and to remove air
bubbles.
21. Clean each solution with ZipTip pipette tips as follows.
μ
22. Put the ZipTip on a 2-20
μ
L scaled pipette and set the volume
L. Wet the ZipTip three times with ZipTip wash solu-
tion and three times with ZipTip equilibration solution (
see
Note 18
).
23. Draw in and out seven times the sample solution. (Do not
pour out the sample solution.)
24. Wash the ZipTip twice with ZipTip equilibration solution.
to 18
μ
25. Draw in 2
L of ZipTip elute solution. Draw in and out three
times and fi nally draw out in a clean Eppendorf tube (PCR
tube).
26. Repeat once
steps 22
-
25
for the same samples. Connect both
the portions and let dry under vacuum.
μ
3.4 LC-MS/MS
Identifi cation
1. Dissolve the sample in 15
μ
L of dissolving solution (5 % ACN,
0.1 % FA).
2. Load 4-10
L of sample (according to the instrument sensitiv-
ity) onto an HPLC chromatography system equipped with a
reversed-phase C18 column (
see
Note 19
).
3. Elute the column at 20 °C with 5 % of mobile phase B in A for
2 min and then over 40 min with a linear gradient of B in A
from 5 to 45 %. Finally elute the column with 45-95 %
gradient of B in A over 5 min before re-equilibration.
4. The eluent from the column is introduced in the electrospray
ionization source of an MS/MS instrument operating in pos-
itive ion mode (
see
Note 20
).
μ
