I performed a phosphopeptide enrichment using TiO2 beads, which worked well, but because of the very limited sample I had, I could not keep a fraction before hand to use for proteomics to compare with the phosphoproteomics. However, I do have the supernatants from my enrichment step which should contain all the non-phosphorylated peptides. Is there a good protocol for removal of DHB so that I can run the non-phosphorylated peptides on my LC-MS?
Hi Christopher,
Why not perform a HILIC cleanup using spin tips (from Glygen, Waters or somewhere else) - this should retain your peptides and get rid of the DHB. Then elute the peptides with a high aqueous solution ready for LC-MS.
Good luck.
Peter
HILIC could work but be careful when comparing phospho vs non-phospho due to 1) obviously ionization efficiencies of phospho are lower thne non-phospho so can not do direct comparison and 2) not all your non-phosho is present in the FG step from the TiO2 enrichment since some will be eluted only in the 2x ACN/TFA washes, so your non-phosho is likely to be under-represented
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