Better would be to use some very good phosphatase, then separate the
phospho from unphospho by ion exchange or iron chelation (which binds
phosphopeptides).
Try lambda phosphatase sold by New England Biolabs (Cat. No. 753)
Ion exchange of phosphopeptides is pretty straight forward, a good HPLC
column, or even a low pressure column on the bench would work fine. For
iron chelation chromatography, we use a variation of the method in this
reference:
Nuwaysir LM, Stults, JT (1993) Electrospray ionization mass spectrometry of
phosphopeptides isolated by on-line immobilized metal-ion affinity
chromatography J. Am. Soc Mass Spectrom 4:662-669
The variation is that we sometimes need a different elution buffer to pop
the peptides off (Elute phosphopeptides with 0.1% Ammonuim Acetate pH 8
(with dilute Ammon. Hydroxide) supplimented with 1 M potassium or sodium
phosphate pH 8 (by appropriate mixing of mono- and dibasic salts) to a
final concentration of 50 mM.), but you want to collect the unbound,
unphosphorylated peptides, so you just need to make sure you have enough
resin to not saturate with the amount of phosphopeptide you have present
(or do a couple of passes--make sure you recharge the resin with iron
before doing second pass).
Katheryn Resing