IMGT and Antibody Protein Sequencing

What Is IMGT?

IMGT is the international ImMunoGeneTics information system for immunoglobulins or antibodies. Understanding the sequence of your antibody is an important, and often critical step in antibody research. IMGT provides a useful reference point that can be helpful when sequencing your antibody. But it is important to know how to treat the variable regions, as these may be very different than any IMGT antibody sequence.

Options for Antibody Sequencing and IMGT

To determine the sequence of your antibody, you have two main options:

  1. If you have access to the cell-line/hybridoma, you may use DNA sequencing techniques to determine the antibody sequence
  2. If you just have an antibody sample or you wish to understand post-translational modifications, phosphorylation, glycosylation, etc. – you can use antibody de novo sequencing (a.k.a. Antibody Protein Sequencing) techniques to sequence your antibody

In both cases, IMGT can be used as a reference.

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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.

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Applications of IMGT after Antibody Protein Sequencing

  • Identifying an antibody sample – found something in the freezer and not 100% sure what it is? Sequence the antibody directly from the sample, then search IMGT for a match
  • Antibody binding affinity troubleshooting – if your antibody is not behaving as expected, try antibody protein sequencing to determine the actual sequence of the antibodies in your sample, then use bioinformatics tools and IMGT to examine the CDR regions
  • Antibody annotation – The tools provided at IMGT can be used to annotate the antibody sequence obtained from antibody protein sequencing.