Protocol for Quantitative Proteomics of Cellular Membranes and Membrane Rafts - LC-MS/MS in Proteomics: Methods and Applications - page 246

tion across the length of a pH test ministrip. If required,
adjust the solution to pH 3.0 or less using 10% formic acid.
This can frequently be achieved with a final concentration
of 0.1-0.2% formic acid added to the 500 μ L reconstituted

-labelled peptide solution.
3. Prepare 15

iTRAQ R

1.5 mL centrifuge tubes each containing 0.5
mL salt fractionation solutions ( see Table 14.1 ) .
4. Assemble the SCX cartridge device and prime the SCX
cartridge with 2 mL SCX loading buffer using a 0.5 mL
glass syringe equipped with a blunt 25 gauge needle.
Flow the solution through the cartridge at

×

∼ 1 drop per

second.
5. Load the reconstituted peptide solution onto the SCX col-
umn at a flow rate of 1 drop per second. Return the effluent
back into the tube originally containing the reconstituted
peptide solution and mix by aspirating briefly with the glass
syringe, then load the solution onto the SCX column again
to ensure complete sample loading.
7. Elute peptides from the SCX column using a step salt gra-
dient by injecting the 0.5 mL salt fractionation solutions in
order of low salt to high salt, i.e. 0-350 mM, collecting the
eluent from each fraction in a 2 mL centrifuge tube.

Table 14.1
Preparation of salt fractions for SCX chromatography

Volume Load
buffer (

Volume Elution
buffer (

Fraction [KCl] mM µ L) µ L)

0(wash) 0 500 -

1 30 457 43

2 50 429 71

3 70 400 100

4 80 386 114

5 90 371 129

6 100 357 143

7 110 343 157

8 120 329 171

9 130 314 186

10 140 300 200

11 150 286 214

12 175 250 250

13 225 179 321

14 350 0 500