Dear colleagues,
Following SCX, we've performed manual desalting of the individually-separated iTRAQ peptide fractions before LC-MS/MS.
We used the commercially-available C18 Pipette Tips for the peptide desalting. Nevertheless, albeit we've increased the number of "rinsing" steps, still we found it difficult to thoroughly remove all the salts (and the performance of our nano-LC column appeared to have compromised / clotted due to the salts).
Would you please share with us your experience - to thoroughly perform peptide desalting after SCX-separation?
Thank you very much.
Hi Kay,
Are you using a trap column to load sample onto your analytical nanoLC column? If so, you could consider performing an extensive wash of the sample after loading (under LC starting conditions), prior to gradient start and switching to the analytical column.
I find that gel filtration-based clean ups always increase sample volumes quite extensively, which normally isn't the best for subsequent nanoLC analyses.
Hope this helps!
You can pretreat/desalt your samples with Sephadex G-15 (if they still make it).
Hi Kay,
Are you using a trap column to load sample onto your analytical nanoLC column? If so, you could consider performing an extensive wash of the sample after loading (under LC starting conditions), prior to gradient start and switching to the analytical column.
I find that gel filtration-based clean ups always increase sample volumes quite extensively, which normally isn't the best for subsequent nanoLC analyses.
Hope this helps!
Hi Kay,
If you have peptide amounts ~>5 microgrammes, you should be using Sep-Pak columns. These columns are very easy to use and have capacity and performance to provide all that you need for iTRAQ derivatized peptides.
Here is a link detailing very through description of usage of SepPak cartridges:
Good luck!
Hediye
http://www.biotech.cornell.edu/sites/default/files/uploads/Documents/Proteomics_protocols/Protocol%2009_SPE%20cartridge%20desalting.pdf
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