The big problem is that the phosphopeptides can get lost in the mess of
unphosphorylated peptides. This is particularly a problem when working
with larger proteins. A very high resolution HPLC column, with a fast
scanning mass spec can be helpful.
Or you can try purifying the phosphopeptides out of the mix by iron
chelating chromatogrpahy--you get a few contaminating acidic peptides, but
the problem is much simplified and sequencing becomes much easier. I think
that the signal from phosphopeptides are easily quenched when analyzed in
the complete digest--several times I have seen weak phosphopeptides appear,
when the digest was simplifed. This happens in both maldi and esi.
For iron chelating chromatography, use of radioactive peptides is easier
because then you can make sure you haven't lost someone (but noncovlanetly
associated 32P can be a serious problem), so you might want to run tricine
gels on fractions. I haven't had any problems spraying 32P, most of it
ends up on the interface plate and needle. If you try CNBr digestion,
note that you often get 80 Da adducts on tyrosines (although these will
show a diagnostic isotopic distribution).
A reference for iron chelating chromatography is
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
I would be glad to give you our protocol, if you are interested, but note
only one person in the lab has used it and then only a few times, so it may
not be a robust protocol.
Katheryn Resing