Proteome-Wide Quantitation by SILAC - LC-MS/MS in Proteomics: Methods and Applications - page 192

impede the following analysis by clouding the peptides
originating from the sample during LC-MS/MS analysis.
14. Add 100-200

L of 50 mM ABC and incubate over night
at 37 ◦ C. The volume of ABC also depends on the total
volume of the cubes, use as much as to cover approximately
1 mm above the cubes.
15. The following day spin down the solution in a bench-
top centrifuge and transfer it to a new tube (this solution
already contains some of the peptides from the digested
proteins).
16. To extract the rest of the peptides, add 100

μ

L of 30%
EtOH/3% TFA solution to the gel cubes, incubate for
10 min and pool solution with that from the preceding
step. (Do not mix solutions from the nine parallel samples.)
17. Repeat Step 16.
18. Repeat Step 16 again but instead of 30% EtOH/3% TFA
solution use 100% EtOH.
19. Dry down peptides in a speed-vac, until all EtOH has evap-
orated.
20. Prepare your sample for MS analysis. This step depends on
the mass spectrometry setup and will not be described in
this chapter.
21. Analyse the samples by LC-MS/MS.
22. To evaluate the efficiency of labeling estimate the ratio
between the labeled and the unlabeled peptides for both
arginine and lysine containing peptides ( see Step 12 in
Section 3.2.2 for description of peptide quantitation).
Generally the average incorporation efficiency should be
above 95%.
23. Perform similar evaluation of the degree of Arg6 to Pro5
conversion by estimating the ratio between the peak from
the peptide containing the normal Pro0 and the peak from
the Pro5 peptide which is shifted 5 Da in the mass spec-
trum. As a general guideline the degree of proline conver-
sion should not exceed 5%.
24. Based on the considerations above select the arginine con-
centration which provides close to full incorporation while
still keeping the degree of Pro conversion at an appropriate
low level ( see Note 6 and Fig. 11.1B ) , select the lowest
lysine concentration giving close to full incorporation.

μ

3.2.Proteome-Wide
Quantitative
Proteomics

After having established the optimal labeling parameters one
can proceed to perform a proteome-wide comparative experi-
ment. The section below depicts a typical workflow of SILAC