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Creative Biolabs is glad to show a series of unique de novo antibody sequencing services for research, diagnostic, and therapeutic industries. To overcome the current drawbacks of sequencing based on traditional methods, Creative Biolabs has developed the proprietary Database Assisted Shotgun Sequencing (DASS) technology, which based on our next generation antibody sequencing platform. Our services consist of variable region, variable plus leader region, as well as full-length heavy- and light-chain antibody sequencing for all species, isotypes, and allotypes. Purified monoclonal antibodies in multivalent forms can be sequenced with 100% coverage of the desired regions as well as excellent accuracy. Numerous successful cases from Creative Biolabs have confirmed our qualification to provide antibody sequencing with 100% accuracy and satisfaction guarantee to meet our customers’ needs.
Our world-leading DASS system provides reliable supporting proof for
Fig. 1 De Novo Antibody Sequencing Workflow.
We could use a high-field Orbitrap Elite hybrid MS (Thermo Scientific) with 240.000 resolution and <1 ppm mass accuracy to perform our services. The available fragmentation techniques will include CID, HCD, and ETD. The combined usage of these technologies could ensure the maximum coverage to generate high-quality results for our clients.
Sequence Validation Guarantee
As we are aware that your projects have tight timelines we validate the sequences to ensure that your antibody will be functional with the first attempt.
(1) The masses of the reduced light- and heavy-chain will be determined to validate the determined sequences. The light chain sequence must match within a +/- 1.2 Da range. For the heavy chain, +/- 1.8 Da is allowed. If the determined sequences do not match with the protein mass, you will not be charged. (Please note that this deviation is less than the weight of two protons.)
(2) Each mutation in the antibody will be validated by at least two meaningful spectra. This ensures that your sequence does not contain isobaric exchanges. Do not rely on peptide maps alone. [Isobaric exchanges are exchanges of identical mass like GG=N, YM=FM (Ox). The peptide maps used by some competitors are often not sensitive to this kind of error.]
If we do not reach at least 5x coverage for the complete V(D)J-region, you will not be charged. You can also book our 10x service. We will not stop to sequence unless we have 10x coverage for V(D)J.
Sequencing of the V and J and C Segments by DASS
The V and J and C gene segments of antibodies are available in public databases. However, during the maturation of an antibody, the B cell introduces hypermutations into the sequence to optimize the affinity. Our mapping algorithm is error tolerant and can match the “mutated” peptides to the corresponding germline, reliably.
Due to the high number of peptides, we get sequence information for EVERY peptide 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. As the order of all amino acids is clear, there is no need for time-consuming techniques like Edman sequencing.
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 be sequenced de novo.
Our method generates many overlapping peptides during the fragmentation process, enabling us to sequence very long stretches of unknown amino acids. The high quality of MS/MS spectra in combination with intelligent data mining, allows us to read the CDR3 like a book. The technique is so powerful that we were able to sequence a 20 kDa protein, which had no homolog in the database.
Isobaric Amino Acids
In contrast to other MS-based methods, we can discriminate most isobaric amino acid combinations. E.g.:
Sequencing of Fluorochrome mAbs, IgMs, and other Non-Standard Antibodies
Fig. 2 Sample Sequencing Results.
If you are interested in our DASS technology, please feel free to inquiry us for more details.
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