I like to know how to prepare plant protein sample for iTRAQ? as this technique needs very specific isolation method(no TRIS buffer, no SDS, no beta-Me, no amine, no ammonia etc). If any one have experience in plant protein isolation technique specific for iTRAQ please share.
Hi Avrajit
Personally I found the easiest thing to do is to extract proteins as you normally would and then buffer exchange into 0.5M TEAB which is iTRAQ compatible. I did this using 3,000 Da centrifugal filters, using ~8 mL TEAB to per mL sample. This is probably massive overkill but I wanted to be certain to remove all contaminants. A bonus of this is that you can also concentrate the sample afterwards - either in the centrifugal filters or in a vacuum centrifuge since TEAB can be evaporated off.
Otherwise I've seen papers where people extract in normal buffer, acetone precipitate a volume containing their required amount of proteins and then resuspend in a minimal volume of TEAB. I'm not a huge fan of this as in my hands it is hard to get the pellet to resolubilise/resuspend effectively after precipitation. Others may have had more luck with this.
Hope this helps
Joe
It really depends on what you are looking for. Any buffer that can solubilise the protein your are looking for is all right. Even if it has ß-ME or SDS as you can always precipitate total protein before labelling to clean it and resuspend in the appropriate buffers. I would rather be more concerned in removing the most abundant proteins like Rubisco that will lower your peptide coverage, as it will hide most of the peptides on the MS analysis. You can remove Rubisco by passing the extracts through a Rubisco depletion column (SIGMA) or by fractionating your extract and analyzing fractions that do not contain Rubisco.
Thanks a lot all of you for answering my question. These suggestions are quite helpful for me. Thanks a lot guys.
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