title: Protein isoaspartate methyltransferase-mediated 18O-labeling of isoaspartic acid for mass spectrometry analysis.
abstract: Arising from spontaneous aspartic acid (Asp) isomerization or asparagine (Asn) deamidation, isoaspartic acid (isoAsp, isoD, or beta-Asp) is a ubiquitous nonenzymatic modification of proteins and peptides. Because there is no mass difference between isoaspartyl and aspartyl species, sensitive and specific detection of isoAsp, particularly in complex samples, remains challenging. Here we report a novel assay for Asp isomerization by isotopic labeling with (18)O via a two-step process: the isoAsp peptide is first specifically methylated by protein isoaspartate methyltransferase (PIMT, EC 2.1.1.77) to the corresponding methyl ester, which is subsequently hydrolyzed in (18)O-water to regenerate isoAsp. The specific replacement of (16)O with (18)O at isoAsp leads to a mass shift of 2 Da, which can be automatically and unambiguously recognized using standard mass spectrometry, such as collision-induced dissociation (CID), and data analysis algorithms. Detection and site identification of several isoAsp peptides in a monoclonal antibody and the β-delta sleep-inducing peptide (DSIP) are demonstrated.
authors: Analytical Research and Development, Amgen, One Amgen Center Drive, Thousand Oaks, California 91320, United States.
authors: Min
authors: M
authors: Liu
authors: Janet
authors: J
authors: Cheetham
authors: Nina
authors: N
authors: Cauchon
authors: Judy
authors: J
authors: Ostovic
authors: Wenqin
authors: W
authors: Ni
authors: Da
authors: D
authors: Ren
authors: Zhaohui Sunny
authors: ZS
authors: Zhou
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003701:Delta Sleep-Inducing Peptide / Q000737:chemistry*; D026581:Isoaspartic Acid / Q000032:analysis*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010104:Oxygen Radioisotopes; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000378:metabolism*
chemical_list: D026581:Isoaspartic Acid; D010104:Oxygen Radioisotopes; D003701:Delta Sleep-Inducing Peptide; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase