since you seem to have the protein radiolabelled, identification of the
site might be pretty straight forward. As you mentioned, digest the protein
with trypsin, or endo LysC, and isolate the radioactive peptide by RP HPLC.
>From there you have several options.
If you have a sequencer guru at your site, you can try solid-phase
radiosequencing. The reference for this is: Wettenhall, et al., Meth.
Enzymol. 201, p. 186. You can actually buy kits these days for
immobilization of the peptide onto disks suitable for the sequencer.
Alternatively, if you don't like the idea of solid-phase sequencing, you
can try to convert phosphoserines to S-ethylcysteines and do ordinary Edman
chemistry on your peptide. This, however, works only for phosphoserines, so
you might have to do some phosphoamino acid analysis first (which you have
to do anyway, also with solid phase sequencing). You'll detect the
phosphoserines as PTH-S-ethylcysteines. Pretty good procedure, but requires
substantial amounts of peptide and stoichiometry of phosphorylation.
For mass spec: a simple experiment you can do is to analyze the radioactive
fraction you collected from the HPLC after digestion and see what masses
you can get. Compare the measured mass with the expected masses of all
peptide fragments. If you find a prominent peptide whose mass is increased
by 80 Da, you have a candidate and you work your way further.
With respect to the argument that one needs high stoichiometry of
phosphorylation to pick it up by MS, I agree with Katheryn Resing's comment
that 5 - 10% stoichiometry is sufficient to pick it up and to analyze the
site. For this, see also Schneider et al. (1999) Biochemistry 38, 4620 -
4632.
Best regards
Paul
Paul Jenoe, PhD
Department of Biochemistry
Biozentrum of the University of Basel
Klingelbergstrasse 70
CH-4056 Basel
Switzerland
Tel. +41 61 267 21 68
Fax. +41 61 267 21 48
e-mail jenoe@ubaclu.unibas.ch