Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Conditions for carrying out chemically targeted identification of peptides containing phosphorylated or glycosylated serine residues have been investigated. Ba(OH)2 was used at ambient temperature to catalyze the beta-elimination reaction at 25 degrees C. Nucleophilic addition of 2-aminoethanethiol was performed in both parallel and tandem experiments. The method was demonstrated by the reaction of beta-casein tryptic digest phosphopeptides and an O-glycosylated peptide. Contrary to an earlier report by others, the glycopeptide was found to react with essentially the same kinetics as phosphopeptides. Conversion of four phosphoserines in residues 15, 17, 18, and 19 from bovine beta-casein N-terminal tryptic phosphopeptides 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 was taken to minimize all counter ions that either precipitate barium or neutralize the base. Digestion of the converted peptides with lysine endopeptidase identified all five phosphoserines in the beta-casein tryptic digest. Alternatively, preincubation with base followed by nucleophilic addition of the thiol was found to work satisfactorily. The use of the water-soluble hydrochloride of 2-aminoethanethiol allowed beta-elimination, nucleophilic addition, and desalting to be carried out on a micro C18 reverse phase pipette tip.
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PMID:Reaction of phosphorylated and O-glycosylated peptides by chemically targeted identification at ambient temperature. 1558 26

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.
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PMID:Determination of phosphorylated and O-glycosylated sites by chemical targeting (CTID) at ambient temperature. 1860 43

A chemical strategy has been developed for identifying phosphorylated and glycosylated sites in peptides. Phosphoserine, phosphothreonine, O-glycosylserine, and O-glycosylthreonine residues in the peptides were converted to the protease-sensitive S-2-aminoethylcysteine derivatives by beta-elimination followed by Michael addition of 2-aminoethanethiol.The resultant lysine analogs were cleaved with Achromobacter lysine endopeptidase.The predicted proteolytic fragments were analyzed by mass spectrometry and N-terminal Edman degradation. When acetylation was carried out as a first step, direct N-terminal chemical sequencing of the digests yielded sequences immediately C-terminal to the phosphorylated or glycosylated residues. Hence, assignment of the sites of modification could be obtained from the chemical sequence data or mass data.Thus, the method offers an approach for rapidly sequencing large,multiply phosphorylated and glycosylated peptides derived from post-translationally modified proteins by both mass spectrometry and Edman chemical degradation.
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PMID:Identification of phosphorylated and glycosylated sites in peptides by chemically targeted proteolysis. 1949 88