Is anyone using iTRAQ? - how do you deal with sample complexity? (Feb/11/2009 )

Hi,

I want to use iTRAQ labeling for my samples. But I don't know how will it get labeled to so many peptides in whole protein complex in sample. How do you reduce the complexity of the sample? and if anyone is using it, can you please share the protocol?


Thanks:)

Protein complexity does not impact the label efficiency so u needn't to consider it before labeling. But the complexity should be considered before MS identification or the identification quality will be poor due to high complexity of protein. Generally, the product producer recommend use SCX to pre-fractionation and reduce the complexity of protein sample. All the protocols are available on the website of AB, u may go there to download or I can mail u a copy but ur email?

amj2 on Feb 11 2009, 04:35 AM said:

Hello,

I am planning to use iTRAQ for my experiments also. I know that SCX followed by LC-MS/MS is the recommended protocol. But I have a doubt:

Can I do SCX-HPLC in my institution, then speedVac dry the samples and send it for LC-MS/MS. Because we do not have LC-MS/MS facility in our institution.

Or SCX should be coupled with Lc-MS/MS? In that case till which step I should perform at my end and ship the samples to the vendor??

Please suggest me. This is URGENT!!

Thanks a lot!