I would like to determine phosphorylation sites on a protein that has been
phosphorylated in vitro with P32 (ATP) using a kinase. I was thinking of
digesting the radioactively labeled protein with proteinase followed by
HPLC purification of the labeled peptide (i.e. phosphorylated) and using an
automated protein sequencer (e.g. Procise) determine which amino acid is
phosphorylated. How reliably can I determine the phosphorylation site
using this strategy and are there better methods? Using Mass Spec is
another strategy but it seems that one needs almost saturation
phosphorylation at the amino acid to detect it.
I would appreciate any comments from colleagues who have experience in this
matter. Jay.
Address: Jay H. Chung MD,PhD
NIH
Bldg. 10-7D13
10 Center Dr.
Bethesda, MD 20892-1654
Tel: (301)496-3075
FAX: (301)496-9985