May I know anyone of you that are working on iTRAQ, what kind of peptide fractionation method / brands you used or recommend for prior to nanoLC (eksigent) separations other than OFFGEL? Anyone of you knows whether ThermoFisher's Pierce High pH Reversed-Phase Peptide Fractionation Kit capable for iTRAQ labeled peptides? Because this kit mentioned it is compatible for TMT tags, but I'm not sure whether is it works well with iTRAQ tags peptides.
Thanks in advance!
Hi,
As you're considering an OFFGEL separation, you could also consider ion exchange prior to reversed phase. Given the reactivity of iTRAQ, you might have more luck using anion exchange instead of cation exchange.
With regard to using reversed phase at high pH followed by reversed phase at low pH: I think that will also work with iTRAQ-labeled peptides, although you might lose some resolution as compared to non-labeled peptides, but that shouldn't be too much of an issue.
Good luck!
Hi there,
In general you can use any kind of fractionation for iTRAQ-label peptides the only difference will be some shift on the retention times accordingly to the method of separation used for example if you do SCX fractionation the peptides will elute at later salt concentrations.
And, yes, you can use the Kit from Pierce, as the chemistry of TMT and iTRAQ are quite similar.
One more think to consider as well, it's that the iTRAQ-label peptides will elute slightly later on the reverse phase that is connected to the Mass Spec.
I hope that this is useful, and good luck with your experiment
Hi Ng,
Martin is absolutely correct - any kind of peptide fractionation should be applicable to iTRAQ as well as TMT labeled peptides. One thing that may sway your decision is if you are specifically after phosphorylated peptides in which case, chromatographic fractionation such as HILIC may be a better choice. Otherwise, in generatl, high PH fractionation seems to results in very good resolution of peptides.
Good luck.
Hediye.
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