Protein Isoaspartate Methyltransferase-Mediated 18O-Labeling of Isoaspartic Acid for Mass Spectrometry Analysis

• Published in: Analytical chemistry (Washington) 2012-01, Vol.84 (2), p.1056-1062
• Main Authors: Liu, Min, Cheetham, Janet, Cauchon, Nina, Ostovic, Judy, Ni, Wenqin, Ren, Da, Zhou, Zhaohui Sunny
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical chemistry; Chemistry; Chromatography, High Pressure Liquid; Delta Sleep-Inducing Peptide - chemistry; Exact sciences and technology; Isoaspartic Acid - analysis; Mass Spectrometry; Molecular Sequence Data; Oxygen Radioisotopes; Protein D-Aspartate-L-Isoaspartate Methyltransferase - metabolism; Spectrometric and optical methods
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac202652z

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 18O 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 18O-water to regenerate isoAsp. The specific replacement of 16O with 18O 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.