Both iTRAQ tags and DiGE CyDye have an NHS reactive group that reacts with primary amines. iTRAQ tags react with the epsilon amino group on lysine and the N terminal group of proteins where as CyDyes react with only the epsilon amino group on lysine.
Why is there this difference when both have the same reactive group, is the addition of the CyDye reagent or the isobaric tag to the NHS group causing this?
Lawrence,
NHS-reactive groups should react with both amino groups in both cases. However, the epsilon amino group has a higher pKA value. So, if you use a buffer that does dissociate the epsilon, but not the alpha amine, you can get preferential modification of the epsilon amine.
In our experience though, this is difficult to accomplish, since neighboring amino acids may also have an influence on the local pH of a peptide.
Bottom line is: both amines react and the closer you inspect it (e.g. by mass spectrometry) the more you will detect both reactions. If you just rely on the color from your CyDye reagent, you may not even notice that it has reacted with both amines.
Lawrence,
NHS-reactive groups should react with both amino groups in both cases. However, the epsilon amino group has a higher pKA value. So, if you use a buffer that does dissociate the epsilon, but not the alpha amine, you can get preferential modification of the epsilon amine.
In our experience though, this is difficult to accomplish, since neighboring amino acids may also have an influence on the local pH of a peptide.
Bottom line is: both amines react and the closer you inspect it (e.g. by mass spectrometry) the more you will detect both reactions. If you just rely on the color from your CyDye reagent, you may not even notice that it has reacted with both amines.
I agree with Sonja's answer. Probably the literature says that CyDye reacts with epsilon amino group, because it was originally designed for labeling of intact proteins. Here, you have a molar excess of epsilon amino groups. Besides, the N-Terminus is often modified, hence unreactive, in proteins.
Besides the peptide iTRAQ there is also a possibility to label on protein level. Definitely the buffer condition is very important here.
Please see the available kits at
http://www.sciex.com/products/standards-and-reagents/iTRAQ-Reagents.xml
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