Separation of tryptic phosphopeptides of ribosomal origin by reversed-phase high-performance liquid chromatography

Phosphorylation sites for cyclic AMP-dependent kinase in ribosomal proteins and their synthetic analogues were converted to tryptic phosphopeptides and analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) using gradients of acetonitrile in water and 0.1% trifluoroacetic acid. Tryptic variants differing by only NH2-terminal basic amino acid residues or phosphoryl groups were not always well resolved under these conditions. The different phospho forms could be resolved by RP-HPLC in phosphate buffers at pH 7.0. A combination of gel permeation chromatography, RP-HPLC and thin-layer cellulose mapping was found to be the most effective strategy for the absolute purification of tryptic phosphopeptides from crude tryptic digests.