Cleavable ICAT

[csang]

Hi,

I am currently using the cleavable ICAT from ABI for comparative analysis of a bacterial pathogen. I'm not sure if this is a problem for anyone but it sure gives me werid results.

The last step of the labelling reaction requires cleavage of the biotin tag using TFA followed by complete drying down using speedvac to remove the TFA??. I realised the dried peptide seems to be hard to dissolve and it tend to give a few broad UV/TIC peaks during LC MS runs. Did anyone using ICAT observe this and what are your solutions?

Thanks in advance!

Ching Seng Ang

[Einar Ponten]

I think this is a so complicated reagent that you have to address this question to the people at ABI. I actually don't beleive that many here know what "icat" stands for.

[Kostas Petritis]

Csang,

Normally you shouldn't have any problems resolubilizing your peptides. At that point you should had a very clean sample containing mainly cysteine containing peptides.

What might have happened is that your digestion in the earlier steps did not work very well so you still have some proteins at that point that are difficult to resolubilise. That would explain also your broad peaks.

Also after digestion you might want to clean up your sample with SPE before doing the rest of the chemistry.

Some more questions... do you use the ABI reagents or do you make your own TFA solution (if yes, you shouldn't as you do not know what else they might have added or what is the exact concentration of TFA they use). Finally, after we cleave, we dilute 10 fold before we dry down with the speedvac.

Hope the above helps,

Kostas

[csang]

Dear Kostas,

Thank you for your suggestions. I'm currently using the reagents provided by ABi except the solubilisation reagent (using 6M urea, 0.05%SDS in 50mM Tris) as the recovery of protein seems better after acetone precipitation as compared to just 0.1% SDS alone.

I have thought of doing a SPE before all the clean ups, but after considering the low amount of protein (200ug total) I thought I would give it a miss. Did you carry out SPE before the SCX and Avidin steps?

The other thing I'm not sure about is the trypsin provided, whether it is modified since it only indicate TPCK treated. I think I will drop ABI an email to check on this.

Thaks again and best regards

Ching Seng