The iTRAQ reagent is very sensitive to water so that is why after taking the iTRAQ reagent vials out of the fridge we need to let it reach to the room temperature to avodi the water in air condensation. But the peptides are resuspended in 0.5 M TEAB water solution. There is water! AT this time the iTRAQ is not afraid of the water?????? I can not understand.
@Robert Ihnatko ，yes, almost I understand what you means. In the way of your explanation, the iTRAQ molecule prefers the amine to the water to react with when water exists in the reaction system and that is why we need a sufficent amount of peptides and peptide concentration in the whole reaction liquid system, because the enough amlount of peptide in the TEAB water solution will be helpful for the iTRAQ reaction preference. Thank you very much for your help.
Hi Ming,
Just to add what Robert mention, The iTRAQ reagent are design to react with primary amine groups at high pH that's the reason why you use TEAB, to give a pH of 8.5; and the iTRAQ reagent won't react with the buffer. Second, in order to dissolved the peptides you need an aqueous solution (TEAB buffer) but the final concentration of the organic in the reactions is around 70% organic (Ethanol, isopropanol), the iTRAQ reagent is dissolved in one of these solvents.
Finally, there is an excess of the reagent (iTRAQ) compared to the peptides, this is in the range of 100 or even more -fold excess of this compared to the peptides. The reaction is favour to the label of the amine groups than its hydrolysis (this is minimal), just in the last step you neutralise this excess adding hydroxylamine.
I hope that this help you understanding the iTRAQ reagent
On top of that I found out that ITRAQ can react slowly even with OH residues if they are enough nucleophilic, there are users note about it . You can try to find them on goggle.
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