Discovering the structures and functions of antibodies in living organism is an important tool in understanding cellular processes, which allow drugs that target specific metabolic pathways to be invented more easily.Antibody sequencing is a technique that determines the amino acid sequence of an antibody. Discovering the structures and functions of antibodies in living organism is an important tool in understanding cellular processes, which allow drugs that target specific metabolic pathways to be invented more easily.
This next generation antibody sequencing technique has now become a routine measurement that can be completed within 10 working days for IgGs. The method is applicable for all antibody formats, namely IgMs, fluorochrome conjugates, immobilized antibodies and mixtures. Creative Biolabs, an emerging qualified service provider in this field, provides researchers their unique technique in antibody sequencing.
With the development of biotechnology, scientists of Creative Biolabs have developed a novel strategy to determine the complete sequence of an antibody with unparalleled speed and accuracy. The technique which combines all the advantages of existing approaches is named "Database Assisted Shotgun Sequencing" (DASS).
In the first step, the antibody is fragmented to peptides by a special technique, which generates up to 5000 different peptides per chain. This set of peptides will be analyzed by high end mass spectrometers to generate extensive sequence information. MS/MS spectra are then de novo sequenced by the latest algorithms and matched against a database with related sequences. Special "in house" data mining tools allow Creative Biolabs to extract the sequence information from ten thousands of MS/MS spectra within hours.
De Novo Sequencing of the CDR3 Region
While the CDR3 of the light chain is mostly encoded by the germline sequences, the CDR3 of the heavy chain is usually not available in databases. It is encoded by the so called D-segments, but these are modified by nucleases and terminal transferases. Typically, only 1-4 AA of a D-segment remain in the matured antibody. The rest of the D-segment is "artificial" and has to go through phage display techniques. Their method generates many overlapping peptides during the fragmentation process, enabling them to sequence very long stretches of unknown amino acids. The high quality of MS/MS spectra in combination with intelligent data mining, allows them to read the CDR3 like a book. The technique is so powerful, that they were able to sequence a 20 kDa protein, which had no homologue in the database.
Sequencing of the V and J and C segments by "Database Assisted Shotgun Sequencing"
The V and J and C gene segments of antibodies are available in public databases. During the maturation of an antibody, the B-cell introduces hypermutations into the sequence to optimize the affinity. Creative Biolabs's mapping algorithm is error tolerant and can match the "mutated" peptides to the corresponding germline reliably.
Because of the great number of peptides, Creative Biolabs is able to get sequence information for ever bond in the antibody. Typically, 20-70 different MS/MS spectra are generated for each amino acid (AA) position. Hence, even the hardest sequences of proline and arginine rich peptides can be resolved.
Learn more about de novo sequence.
About Creative Biolabs
Creative Biolabs is specialized in providing custom biotechnology and pharmaceutical services that cover the full scope of biotechnology needs of early drug discovery and development, including antibody production , membrane protein preparation, etc.