COMPLETE DE NOVO SEQUENCING OF MAB
The service for the complete de novo sequencing of mAb and other proteins offered by Gemini Bio is based on a proprietary protein sequencing technology that we developed over many years. We guarantee a result with a 100% sequencing coverage of the entire mAb molecules, a better than 99.5% accuracy and distinct assignment of Leu/Ile residues.
We also offer an optional confirmation service to confirm the accuracy of the variable regions of the sequenced mAb. In this validation process, we express HV and LV region O in E. coli, and use LC/MS/MS peptide mapping to compare each variable region peptide from original and expressed proteins. We guarantee a 100% correctness of the mAb sequence after this confirmation process. Since we use E. coli as the expression host, the confirmation process is often much faster and lower cost than mammalian host expression. Since the sequence confirmation is based on MS and MS/MS of each peptide instead of ELISA assay, the sequence fidelity is much higher.
The complete mAb de novo sequencing is often a very complex process, involving more than ten different proteolytic digestions, several steps of chemical derivatizations, NanoLC-ESI-MS/MS data acquisition, bioinformatics analysis with more than a dozen proprietary softwares, the manual spectra assignment, etc. In general the process contains the following steps:
Proteolytic digestion and chemical derivatization.
LCMS data acquisition.
Database search and peptide de novo sequencing.
Draft sequencing assembling.
Sequence gap filling.
Sequence error screening.
Our mAb de novo sequencing technology can also be applied to other proteins. However, changes in the process are often required based on the nature of the protein.
Sample requirement: Since more than ten different proteolytic reactions are carried out in the sequencing process, we prefer to have > 1mg mAb protein for each project, although ~500ug mAb sample is acceptable. A better than 90% purity is required. The main concern about the quality of a sample is immunoglobin contamination since there is no easy way to separate contaminating antibodies from the mAb of interest and it is not easy to determine if a peptide is from a contaminating protein or from the mAb of interest. The major source of such contamination is the foetal calf serum in cell culture media.
It often takes about four weeks to sequence one mAb sample.