Antibody De Novo Sequencing: Revealing Amino Acid Sequences

Antibody De novo sequencing refers to the analytical process of deducing the amino acid sequence of an antibody and discovering any related post-translational modifications without prior knowledge of the DNA or protein sequence.

 

The sequence information of an antibody is crucial for understanding the structural basis of antigen binding, and enables the antibody to be used as a therapeutic and research tool. Sequencing of monoclonal antibodies aids in the development of biopharmaceuticals and diagnostic kits. In particular, primary antibody sequence analysis is the basis for antibody development and humanized antibody drug development. However, due to the variation and modification of antibody products, the sequence information of most antibodies is often not included in available databases. De novo sequencing technology employs advanced data processing algorithms for sequence analysis, offering superior advantages compared to traditional methods that rely on database searches. The use of de novo sequencing methods allows for accurate analysis of antibody sequencing, without the need for database searches.

 

In a typical antibody de novo sequencing workflow, a set of different enzymes are typically used to digest the antibody (one at a time or in combinations), including the most commonly used enzyme: trypsin, and several others such as Glu-C, Asp-N, if required, the heavy and light chains of the antibody can be separated before enzymatic digestion. The digested peptides are then analyzed by LC-MS/MS. Finally, the most advanced computational algorithms are used for data analysis, effectively deducing the amino acid sequence of the digested peptides and assembling overlapping peptides.