Antibody protein sequencing is simply defining the sequence of amino acids of an unknown antibody. The use of mass spectrometry in de novo sequencing of antibody protein has made it possible to perform this without having the cell lines available or knowing the DNA sequence beforehand.
The mass spectrometry measurements of the protein molecule serves as the base for the sequencing technique. It requires only 0.2 mg of the protein. The time between analysis and reporting has been cut to weeks. The detailed report includes both heavy chain and light chain sequences, the associated data which supports the reported sequence, and other comments on the protein molecule.
Key Benefits of Antibody De Novo Sequencing
The benefits of using de novo sequencing on an antibody includes:
Reproduction of an Antibody Without the Producer Cell
If a hybridoma which yielded a particular antibody is lost or not available, the antibody itself can still be reproduced by recombination, using just 0.2 mg of monoclonal antibody protein. If even this much is not possessed, other means may still be possible.
End-To-End Sequencing of an Antibody
The use of high resolution mass spectrometry techniques with optimized settings, and the proprietary software for de novo sequencing of the antibody, means that the whole protein can be sequenced from the n-term to the c-term including every amino acid.
Unsurpassed Accuracy
Each amino acid is correctly identified in the sequence because of the strong signal peak signatures obtained in multiple mass spectrometry readings of separate peptides.
Range of Applications
The mass spectrometry-based technology can be used to sequence all types of antibodies, whether IgA, IgM, IgG, IgY, Fab, ScFv, and so on, from all types of animal species including mice, rats, rabbits, and hamsters, apart from humans. It has even been used to sequence contaminated or conjugated antibodies or those which have different light chains in a pair.
The Pursuit of Perfection
The aim of the technology is perfect mass spectrometric protein sequencing. This has led to unceasing efforts to produce an optimized workflow and software to make it possible to sequence every protein, no matter how difficult, as well as to maintain the highest quality standards.
Report Everything
The use of this antibody de novo sequencing has often led to the detection of unexpected differences in sequencing or novel glycosylation sites on the protein molecule, which are not picked up by DNA sequencing.
High Throughput
The mass spectrometry approach to de novo antibody sequencing is useful with just one or hundreds of antibody molecules awaiting sequencing.
Sample Requirements
In a typical situation, the technology requires 0.2 mg or more of monoclonal antibody of at least 95 % purity. Following analysis, the deliverable consists of a detailed report giving the complete sequences of both the heavy and light chains, with associated data to support the results, and any other useful comments such as the observation of unexpected variations of the antibody. The time required is typically 1-2 weeks from the receipt of the sample.
Other Antibody Sequencing Techniques
Several other techniques have been made use of to accomplish antibody sequencing:
Hybridoma Sequencing
In this technique, the cDNA that encodes the variable heavy and variable light (VH and VL) domains of the hybridoma cell line is sequenced. This is an established technology, and is the most cost-effective provided the cell line secreting the antibody of interest is available.
Edman Sequencing
This technology has been available for many years, and can be used for a direct sequence determination if the protein is 50 amino acids long or less. It handles low throughputs only.
Intact Mass Analysis and Peptide Mapping
These tools are not identical with sequencing of antibodies by mass spectrometry but can confirm the accuracy of the determined sequences of the antibody protein. Intact mass analysis tells us whether the protein molecular weight is identical with the experimental observation at the level of the protein. Peptide mapping helps us know whether the precursor peptides are as expected. However, these techniques are restricted in their reach, and cannot detect the individual amino acids themselves, which is why they are not useful in sequencing per se.
About Rapid Novor Inc
Rapid Novor Inc. is the world's leader in antibody protein sequencing technology. Specializing in the field of mass spectrometry-based proteomics, the team has developed the technology to directly sequence antibody proteins without needing to access the producing cell line.
As a University of Waterloo spin-off, the company is building its technology portfolios based on the twenty years of scientific research and inventions from Dr. Bin Ma, co-founder and chief scientist. The company's REmAb™ antibody protein sequencing service has allowed for the accurate sequencing of any given antibody proteins on a routine basis. The company's WILD™ service is the first commercially available service that can accurately distinguish the isomeric Isoleucine and Leucine using mass spectrometry.
Rapid Novor Inc's mission is to advance life science for better human health with next generation protein sequencing.
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