Protein Isoaspartyl Methyltransferase-Mediated ~(18)O-Labeling of Isoaspartic Acid for Mass Spectrometry Analysis

摘要

Methylation via PIMT specifically occurs at isoAsp, not Asp, to generate the corresponding methyl ester that is labile and converted to unstable succinimide intermediate. Hydrolysis of succinimide in ~(18)O-water forms ~(18)O-isoAsp (major product) and ~(18)O-Asp (minor product). Complete hydrolysis was evidenced by the disappearance of the methyl ester and succinimide peak. The isotopic patterns also provided additional support to high ~(18)O-labeling efficiency. The specific ~(18)O-labeling at isoAsp results in a mass shift of 2 Da leading to automatically and unambiguously detect and identify site of isoAsp peptides in IgG1 and beta-delta sleep-inducing peptide (DSIP).