Hi all,
This is regarding using Reverse Phase High pH Fractionation Cartridges being provided by Thermo.
We are planning to use these cartridges for Fractionation before submitting iTRAQ labled samples to Mass Spec. Has anyone used these cartridges with iTRAQ samples. The Sheet provided by Themo says it can be ideally used fractionate any complex mixtured sample, however the manual shows separate protocols for unlabled and TMT labeled samples; which creates a dilemma in mind that will it work fine with iTRAQ lablled samples.
Theroritically, I agree that one can use this cartridges for sample lablled with iTRAQ reagent as well. However I wanted to know following
1. has anyone tried out it
2. did it work well and
3. if yes did you have to modify the elution or the same as provided in sheet worked well.
4. if you modified, can you share modifications done in protocol.
Thanks.
Joel
I must say that ODS silica gel should be used in acidic phosphate buffer (pH 2.0) conditions (please see files; Monch Protein separation and Lysozyme by RP-HPLC). Silica gel is labile to alkaline eluents.
Please, handle the silica gel without using TFA. Acidic TFA solution generally gives low recovery of peptide or protein as compared to acidic phosphate buffer (pH 2.0) (our unpublished observation).
Since MS is not a quantitative detector at all, the acidic phosphate buffer seems not to reduce the signals (my personal and unpublished observation).
I would like to recommend that fractionation of peptide by the above RP-HPLC method, and fractioned peak should be quantitatively identified by Edman degradation method (please see file about PDMD (protein/peptide-direct-microsequencing-deciphering) method; HepG2 fucoidan).
I also would like to say that "slow but steady wins the race".
You will obtain hysrolysis of the stat. phase and consequent loose of efficiency.
Hello thanks for your reply, but this kit works really fine in separating complex peptide mixture generated by Digestion of proteins with any known enzyme. People have reported it several publications. but I haven't seen it being used along with iTRAQ reagent.
so my question is pertaining to its feasibility in using it for fractionation of iTRAQ labeled samples.
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