Procedure for Antibody Protein Sequencing
Antibody Protein Sequencing is generally achieved in a bottom-up manner by the following steps: (1) Enzymatically digest the protein into shorter peptides. (2) Conduct mass spectrometry measurement to produce the tandem mass spectrometry (MS/MS) data for the peptides. (3) Derive the sequence of each shorter peptide from the MS/MS data (a.k.a. De novo peptide sequencing). (4) Assemble the peptides sequences back to the long protein sequence.
IMGT and peptide mapping
After the Liquid Chromatography -Tandem Mass Spectrometry (LC-MS/MS) measurement of the peptides, the germline sequences in IMGT database can be searched to map the peptides in the constant regions of the antibodies. This method will help identify the host species, isotypes, and subclasses of the antibody. In addition, sometimes the gene and locus of the antibody may be determined this way. However, this method is not applicable to the hypervariable regions of antibodies. Peptide mapping cannot be used as an antibody sequencing method.
IMGT and de novo antibody protein sequencing
Antibody de novo sequencing (a.k.a. Antibody Protein Sequencing) methods can produce whole antibody sequences with a high degree of confidence and without need for a DNA reference sequence. Read more about how Antibody Protein Sequencing works. Once an antibody has been sequenced in this way, its sequence can be searched against IMGT to find homologous antibodies.