De novo assisted AFB1-Specific monoclonal antibody sequence assembly and comprehensive molecular characterization
peer-reviewed · Analytical Biochemistry · 2022
| Date | 2022-11-01 |
| Type | peer-reviewed |
| Venue | Analytical Biochemistry |
| Publisher | Elsevier BV |
| Contribution | downstream-application |
| DOI | 10.1016/j.ab.2022.114883 |
| Citations (OpenAlex) | 3 |
Abstract
Despite their widely used and access as biological reagents in analytical methods, the detailed structural features for most of the antibodies were rarely known. Here, a new antibody for AFB1 with high specificity in constructing ELISA was studied in detail. The molecular structure and modification were elucidated mainly by nano-electrospray ionization mass spectrometry. The mass experiments, including MALDI-TOF MS, revealed complete and specific fragments, including antibody molecular weight, peptides, glycopeptide, and N-glycoform. By proteolytic treatment of pepsin and trypsin and high-resolution tandem-MS, the primary structure of the newly developed anti-AFB1 antibody was assembled by several rounds of Database search process assisted with the de novo results. The antibody CDR annotation and constraint-based multiple alignment tool were used to differentiate and align the sequences. The method uses only two proteases to generate numerous peptides for de novo sequencing. This artificial assembled AFB1-specific monoclonal antibody sequence was validated by comparison with the sequencing results of the immunoglobulin gene. The results showed that this method achieves full sequence coverage of anti-AFB1 monoclonal antibody, with an accuracy of 100% in the CDR regions of light chain and four amino acid mismatch in heavy chain. This simple and low-cost method was confirmed by treating a public dataset. The secondary structure information of intact antibody was also elucidated from the results of circular dichroism spectrum.
Methods and tools
- Anti-AFB1 mAb de novo assisted assembly: Two-protease digestion and de novo-assisted assembly gave full sequence coverage of an aflatoxin B1 monoclonal antibody, with all CDRs correct.
Methods it uses
- PEAKS: Commercial DP-based de novo