The objective of de novo protein sequencing is to reconstruct the amino acid sequence of a peptide/protein given a MS spectrum without reliance on previously established databases. This powerful method is key to characterizing proteins, in particular novel protein sequences.
De novo protein sequencing has drawn much attention with regard to antibody sequencing. Antibodies are essential molecules in our immune system and serve as invaluable tools in the fields of diagnostics and therapeutics. Under standard commercial conditions, antibody production begins once an animal is inoculated with a desired antigen. This drives an immune response that involves genetic hypermutation of plasma cells and rearrangement events that create polyclonal antibodies. Using molecular techniques such as hybridoma generation and single B cell sequencing, researchers can then isolate individual clones (monoclonal antibodies) from the mixture.
Critically, immunizing the original animal with an identical antigen will produce differing batches of monoclonal antibodies — each bearing unique specific activities. In this instance, DNA sequencing could be used to identify matching antibodies; however, as it involves extensive bioinformatic analysis and detailed workflows, researchers typically select an alternative approach.
De novo protein sequencing using mass spectroscopy is a more convenient way to determine the monoclonal antibody code, which can then be used for recombinant antibody expression to ensure reproducibility. When used in conjunction with complementary biophysical and biochemical methodologies, this method also allows for elucidation of the structure, avidity and PTMs of antibodies. In addition, an important feature of de novo protein sequencing is that it avoids random or biased sequence introduction from homology-based searching of databases1. For this reason, it is a key tool for the process of antibody validation.
From a practical perspective, de novo protein sequencing is especially useful as it can be used once an antibody-producing cell line has ended, when DNA is unavailable, or when the production animal has died. Accordingly, MS-based de novo protein sequencing is an excellent tool for commercial and non-commercial antibody research and development.