A SELECTIVE PRECIPITATION PURIFICATION PROCEDURE FOR MULTIPLE PHOSPHOSERYL-CONTAINING PEPTIDES AND METHODS FOR THEIR IDENTIFICATION

Multiple phosphoseryl-containing sequences of proteins stabilize amorp hous calcium phosphate and have been implicated in the regulation of b iomineralization, protein structure, and enzyme activity. To facilitat e studies on the identification and characterization of multiple phosp hoseryl-containing sequences of proteins we have developed a simple an d efficient purification procedure involving precipitation of Ca2+/eth anol-induced aggregates of the multiple phosphoseryl-containing peptid es from enzymic digests. The multiple phosphoseryl-containing peptides of a tryptic digest of casein were selectively precipitated using Ca2 + (20 mol/mol protein) and 50% (v/v) ethanol at pH 3.5, 4.6, and 8.0. The individual peptides of the precipitates were purified using anion- exchange fast-performance liquid chromatography and reversed-phase HPL C and then identified by solid-phase sequence analysis and amino acid composition analysis after vapor-phase hydrolysis. Prior to sequence a nalysis the phosphopeptides were covalently coupled to arylamine membr anes and the phosphoseryl residues converted to S-ethylcysteinyl resid ues by calcium-ion-catalyzed beta-elimination in the presence of ethan ethiol. The modified peptides were sequenced using an Applied Biosyste ms Inc. automated protein sequencer fitted with a membrane cartridge. Only peptides containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)- w ere precipitated by Ca2+/ethanol at pH 3.5. The pH 4.6 precipitate con tained all the cluster peptides plus two diphosphorylated peptides con taining -Ser(P)-Glu-Ser(P)- and -Ser(P)-Thr-Ser(P)-. At pH 8.0, a mono phosphorylated peptide containing -Ser(P)-Glu-Glu- was also present in the precipitate with the diphosphorylated and cluster peptides. The r ecoveries of the peptides in the pH 8.0 selective precipitate ranged f rom 83 to 95% of that present in the hydrolysate. All of the phosphope ptides in the casein tryptic hydrolysate were selectively precipitated at pH 8.0 by Ca2+/ethanol, except the two monophosphorylated peptides containing -Ser(P)-Ala-Glu- and -Ser(P)-Thr-Glu-, suggesting that, fo r a monophosphorylated peptide, the minimal motif for Ca2+/ethanol-ind uced precipitation is -Ser(P)-Glu-Glu-. (C) 1994 Academic Press, Inc.