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.
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008099:Liver / Q000032:analysis; D008297:Male; D010748:Phosphopeptides / Q000302:isolation & purification*; D011494:Protein Kinases / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D012270:Ribosomes / Q000032:analysis* / Q000201:enzymology
D010748:Phosphopeptides; D011494:Protein Kinases