36 A Practical Guide to Cancer Systems Biology
- Vortex erectly at room temperature for 1 hour.
- Do not vortex too vigorously.
- Prepare a new 2 mL protein lobind tube. Spin down the labeled peptides
and combine the four samples of labeled peptides to the new tube.- Do not combine the labeled peptides to one of the sample tube to
ensure the amount of peptides of each sample in the combined tube
is equal.
- Do not combine the labeled peptides to one of the sample tube to
- Dry the labeled peptides with centrifugal evaporator.
No need to make the sample too dry. Proceed to the next step when the
labeled peptides become as a small drop of brown liquid.
ZipTip desalting
- For small-scale experiment, desalt the iTRAQ-labeled peptides
directly. - For large-scale experiment, we recommend performing strong cation
exchange (SCX) chromatography after iTRAQ labeling. Dry every
fraction of iTRAQ-labeled peptides with centrifugal evaporator and
desalt each fraction individually.
- Resuspend the dried iTRAQ-labeled peptides with 20− 30 μLof0.1%
TFA. - Adjust the pH of each sample to about pH 2–3 with 10% TFA. Examine
thepHwithpHteststrip.
- Use the P20 pipetman for the following steps.
- Pre-wet the ZipTip by aspirating 50% ACN/0.1% TFA into tip and
dispense to waste on a paper towel. Repeat this step for 5 times. - Equilibrate the ZipTip by aspirating 0.1% TFA into tip and dispense to
waste on paper towel. Repeat this step for 10 times. - Sample binding by aspirating and dispensing the sample for 20 cycles.
- Be sure to aspirate completely to let 20μL of sample pass through the
membrane in ZipTip each cycle.
- Wash the ZipTip by aspirating 0.1% TFA into tip and dispense to waste
on paper towel. Repeat this step for 10 times. - Elute the peptides by aspirating 50% ACN/0.1% TFA into tip and
dispense the eluent (20μL) into a new 2 mL protein lobind tube. - Aspirate and dispense the eluent in the tube for 10 cycles.