34 A Practical Guide to Cancer Systems Biology
- Resuspend the dried peptides in each final peptide tube with iTRAQ
Dissolution Buffer. Combine the peptide solution in each final peptide
tube and the volume of the combined solution should be as calculated in
step 1. - Vortex the sample for several minutes to fully dissolve the peptides in the
buffer. - Examine the pH with pH test strip. The pH of each sample should be
about pH 8.5. - The peptide solution can be stored at− 30 ◦Cforafewdays.
Small-scale experiment: test condition and reproducibility
- The amount of peptides required for iTRAQ labeling is 5μgof
peptides for each sample.
- Measure the concentration of peptides with Pierce BCA Protein Assay
Kit. - Transfer the appropriate amount of peptides to a new 2 mL protein
lobind tube. - The amount of peptides in different samples should be equal for
comparison. Adjust each sample to have equivalent amount and volume
with iTRAQ Dissolution Buffer. - Label the tube with the name of the sample and iTRAQ isobaric tag
number intended to label the sample. - Bring the iTRAQ Reagent vials supplied in the iTRAQ Reagents
Multiplex Kit to room temperature. - Add 70μL absolute ethanol supplied in the iTRAQ Reagents Multiplex
Kit to each iTRAQ Reagent vial. Vortex the mixture for 1 minute and
spin down. - Calculate the amount of iTRAQ Reagent needed to label each sample.
Each iTRAQ Reagent vial is able to label up to 75μgofpeptides.Be
sure that the percentage of ethanol in each sample mixed with iTRAQ
Reagent must be over 50%. - Equal amount of peptides from different samples are labeled by adding
the calculated amout of iTRAQ Reagent 114, iTRAQ Reagent 115,
iTRAQ Reagent 116, or iTRAQ Reagent 117. - Cap and seal the 2 mL protein lobind tube containing sample with
parafilm carefully. - Vortex erectly at room temperature for 1 hour.
- Do not vortex too vigorously.