Biology Reference
In-Depth Information
3. Asses protein quantifi cation using a suitable protein assay kit
(e.g., BCA assay or Bradford) using at least three serial dilu-
tions of your sample with three replicates each and average
quantifi cation values. Check assay compatibility with sample
buffer.
4. Aliquot up to 100
g of protein for labeling. And perform
acetone precipitation as described above.
5. Resuspend completely the protein pellet in 30
μ
μ
L iTRAQ
L of 2 %
(w/v) SDS and if needed, followed by incubation of the pro-
tein sample overnight at 4 °C (
see
Note 4
).
6. Reduce disulfi de bonds by adding 2
dissolution buffer provided in the kit adding 3
μ
L of reducing agent from
iTRAQ kit (50 mM TCEP). Vortex, pulse spin, and incubate
at 60 °C for 1 h.
7. Bring protein sample to room temperature and alkylate by
adding 1
μ
L of cysteine-blocking reagent from iTRAQ kit
(200 mM MMTS). Vortex, pulse spin, and incubate for 10 min
at room temperature.
8. Dilute protein sample by adding 250
μ
L of 100 mM iTRAQ
dissolution buffer to ensure that SDS is lower than 0.05 % and
perform digestion by addition of trypsin at a 10:1 protein:trypsin
ratio (i.e., 10
μ
g protein). Vortex, pulse spin,
and incubate at 37 °C overnight (
see
Note 5
).
9. After overnight incubation, trypsin is added in a 20:1
protein:trypsin ratio (i.e., 5
μ
g trypsin per 100
μ
g protein) to
ensure complete protein digestion. Vortex, pulse spin, and
incubate at 37 °C for 6 h (
see
Note 6
).
10. Protein sample digested is evaporated frozen under vacuum or
lyophilized (
see
Note 7
).
11. Reconstitute peptides in 30
μ
g trypsin per 100
μ
L iTRAQ dissolution buffer.
12. Thaw iTRAQ reagent vials to room temperature and add
70
μ
L of absolute ethanol provided in iTRAQ kit. Vortex dur-
ing 1 min, pulse spin to take all the volume at the bottom of
the vials, and add this solution to each sample. Incubate at
room temperature for at least 1 h (
see
Note 8
).
13. Add to each sample 1 mL of SCX-A buffer. Samples can be
kept at 4 °C if the analysis is to be continued immediately or
stored at −20 °C but not longer than 1 week (
see
Note 9
).
μ
3.2 Sample
Pre-fractionation
1. Samples are pooled in a 15 mL clean tube and each sample
tube is washed out with an extra 100
L SCX-A buffer and
added to pooled material ensuring recovery of all peptides.
2. Make fi nal volume up to 5 mL with SCX-A buffer and check
pH. Ensure that pH is ranged from 2.5 to 3.0 by adding
small amounts of phosphoric acid.
μ
