Determination of phosphorylated and O-glycosylated sites by chemical targeting (CTID) at ambient temperature.

Abstract

In the analytical approach called chemically targeted identification (CTID), peptides containing phosphorylated or glycosylated serine and threonine underwent beta-elimination to produce an unsaturated double bond. Nucleophilic addition of 2-aminoethanethiol to this bond occurred, yielding aminoethylcysteine. Thus, sites containing posttranslational modifications were made susceptible to lysine endopeptidase. Structural information could then be obtained by mass analysis of the proteolytic products. The method was demonstrated by the analysis of beta-casein tryptic digest peptides and an O-glycosylated peptide. Contrary to an earlier report, the glycopeptide was found to react with essentially the same kinetics as phosphopeptides. Conversion of all five phosphoserines in residues 15, 17, 18, 19, and 35 in N-terminal tryptic phosphopeptides from bovine beta-casein were followed by monitoring the time course of the addition reaction. The chemistry proceeded rapidly at room temperature with a half-reaction time of 15 min. No side reaction products were observed. However, care had to be taken to minimize all counterions, which either precipitate barium or neutralize the base. In the case of 2-aminoethanethiol, excess Ba(OH)2 was needed to offset the effect of the hydrochloride. Alternatively, pre-incubation with base followed by nucleophilic addition was found to work satisfactorily. The use of water-soluble thiol allowed the procedure to be carried out in the solid phase, with a micro pipet greatly facilitating sample cleanup.