Automatic Disulfide Bond Assignment Using a1 Ion Screening by Mass Spectrometry for Structural Characterization of Protein Pharmaceuticals

• Published in: Analytical chemistry (Washington) 2012-06, Vol.84 (11), p.4900-4906
• Main Authors: Huang, Sheng-Yu, Hsieh, Yu-Ting, Chen, Chun-Hao, Chen, Chao-Chi, Sung, Wang-Chou, Chou, Min-Yuan, Chen, Sung-Fang
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical chemistry; Antibodies, Monoclonal - chemistry; Biological and medical sciences; Chemistry; Chemistry, Pharmaceutical; Classical genetics, quantitative genetics, hybrids; Disulfides - chemistry; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Genetics of eukaryotes. Biological and molecular evolution; Humans; Ions; Methods, theories and miscellaneous; Molecular Sequence Data; Recombinant Proteins - chemistry; Software; Spectrometric and optical methods; Spectrometry, Mass, Electrospray Ionization; Staining and Labeling; Tandem Mass Spectrometry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac3005007

An automatic method for disulfide bond assignment using dimethyl labeling and computational screening of a1 ions with customized software, RADAR, is developed. By utilization of the enhanced a1 ions generated from labeled peptides, the N-terminal amino acids from disulfide-linked peptides can be determined. In this study, we applied this method for structural characterization of recombinant monoclonal antibodies, an important group of therapeutic proteins. In addition to a1 ion screening and molecular weight match, new RADAR is capable of confirming the matched peptide pairs by further comparing the collision-induced dissociation (CID) fragment ions. With the N-terminal amino acid identities as a threshold, the identification of disulfide-linked peptide pairs can be achieved rapidly at a higher confidence level. Unlike most current approaches, prior knowledge of disulfide linkages or a high-end mass spectrometer is not required, and tedious work or deliberate interpretation can be avoided in this study. Our approach makes it possible to analyze unknown disulfide bonds of protein pharmaceuticals as well as their degraded forms without further protein separation. It can be used as a convenient quality examination tool during biopharmaceutical development and manufacturing processes.