What Is Antibody De Novo Sequencing?

Background of De Novo Sequencing

Proteins that make up biopharmaceuticals are bio-macromolecules that sustain life activities. They are composed of one or more peptide chains, each of which is connected by a certain number of amino acids in a certain order through peptide bonds. The amino acid sequence of a protein constitutes its primary structure, and protein sequencing is the process of determining this sequence. Protein sequencing can provide very useful information, including: identifying proteins; providing information on post-translational modifications (PTMs), which are important for the structure and function of proteins; improving antibody drug development, etc. Therefore, protein sequencing is an important technology in biomedicine.

Currently, the protein sequencing methods on the market can be mainly divided into three types: Edman degradation sequencing, PCR-based protein sequencing, and mass spectrometry-based protein sequencing.

• Edman degradation sequencing: This is an early developed protein sequencing technology that uses chemical methods to degrade amino acids from the N-terminal of the protein in sequence, then uses high-performance liquid chromatography to identify the amino acids. However, this method has certain limitations. It can only sequence the first 20–25 amino acids of the N-terminal and cannot be applied to proteins with closed or modified N-terminals.
• PCR-based protein sequencing: This method utilizes the DNA or RNA expressed in cells for gene sequencing and then converts it to the amino acid sequence of the protein according to the amino acid codon table. However, this method fundamentally cannot fully reflect protein-level information and cannot identify important information, such as post-translational modifications.
• Mass spectrometry-based protein sequencing: This method has higher sensitivity and specificity, and because of its strong qualitative ability, it can analyze the peptide segments enzymatically derived from proteins in a bottom-up manner. It can identify closed ends and provide information about post-translational modifications, with much fewer mismatches. In the past, it was thought that amino acids with close or identical mass numbers, such as lysine vs. glutamine, leucine vs. isoleucine, could not be distinguished by mass spectrometry. However, with the popularization of high-resolution mass spectrometry, and the advancement of fragmentation technologies such as ETD and EAD, these problems have been overcome.

Our optional protein sequencing services:

• Hybridoma Sequencing
• De Novo Antibody Sequencing

Definition of De Novo Sequencing

Protein de novo sequencing technology is the process of analyzing the amino acid sequence of a protein by using tandem mass spectrometry (MS/MS) to obtain ions and calculate the mass of amino acid residues on the peptide chain based on the mass difference between two fragment ions. The biggest advantage of this technology is that it does not need to rely on any database and can analyze the sequences of known or unknown proteins.

Workflow of De Novo Sequencing

The samples were hydrolyzed by multiple enzymes, and the peptides were detected by mass spectrometry. The mass spectra of the collected peptides were analyzed by a de novo algorithm, and the corresponding peptide sequences were obtained. After the comparison and splicing of peptides, the amino acid sequences of the proteins were obtained from the N-terminal to C-terminal of the protein. At present, the platform of protein de novo sequencing can be established by liquid chromatography coupled with high resolution mass spectrometer, which ensures the sensitivity of the identification of low abundance peptide fragments. At the same time, the fragmentation mode of HCD and ETD binding is adopted to distinguish the isomers of leucine and isoleucine.

Fig. 1 Overview of novel antibody discovery approach. (Adrian Guthals, 2017)

Our de novo antibody sequencing services:

• De Novo Biosimilar Monoclonal Antibody Sequencing
• De Novo TCR (T Cell Receptor) Sequencing
• De Novo Rodent Antibody Sequencing
• De Novo Rabbit Antibody Sequencing
• De Novo Chicken IgY Sequencing

Data Analysis

Peptide coverage: The protein samples were digested based on specific and non-specific proteases respectively to achieve 100% protein coverage, produce rich and highly overlapping peptide types, and finally get the high-quality data needed for sequencing.

Fig. 2 General workflow for sequencing of novel proteins via PEAKS based on an electropherogram. (Jianhui Cheng, 2020)

Peptide splicing: Using HCD will produce a series of b/y ions, and the sequence of peptides can be deduced according to these ions' information. However, because many kinds of endonuclease digestion will produce a lot of repetitive peptides, the peptides should be screened. The first step is to select the peptide with high kurtosis, which is actually to judge the fragmentation of the secondary ions of the peptide. However, when the fragment ions are missing, the database search may still be successful. Therefore, in order to improve the accuracy of ab initio sequencing, the method may be combined with different ion fragmentation techniques to produce multiple spectra.

Discrimination of leucine & isoleucine: In the past, Leu and Ile were often considered indistinguishable by MS because their molecular masses were exactly the same. Incorrectly positioned Leu/Ile residues within variable domains, particularly within the CDRs (complementarity determining regions) of an antibody, can result in substantial loss of antigen-binding affinity and antibody specificity. Leucine and isoleucine can obtain c/z ions under ETD fragmentation conditions, and then assist HCD fragmentation to remove some groups based on z ions to form w ions. Leucine removes the propyl group, forming a 43Da loss. Isoleucine removes the ethyl group, forming a 29Da loss. This allows precise discrimination between leucine and isoleucine.

Data Verification

Compared with database search methods, the main limitation of current ab initio sequencing software is the lack of automatic statistical verification. If there is no such automatic verification method, you still need to check the peptide map matching manually to determine which ab initio sequencing results to take seriously.

Molecular weight verification: The basic modification information (oxidation, deamidation, N-terminal cyclization, C-terminal K deletion, glycosylation) can be obtained from the protein molecular weight, so as to verify the correctness of the protein sequence obtained by de novo sequencing.

Protein sequence verification: A new database was established for the sequences obtained by de novo sequencing, and the coverage analysis of the collected mass spectrometry data was performed again, and an almost complete matching result was obtained.

Fig. 3 The matched heavy chain contigs for infliximab after 1^st homologous database search. (Jianhui Cheng, 2020)

Applications of De Novo Sequencing

Protein sequencing technology can detect biomarkers of disease and provide a new way to study cancer and other problems. Proteins produced by cells can act as unique biomarkers in many diseases, such as cancer, Alzheimer's disease, heart failure, and diabetes, and more accurate detection of these biomarkers can help researchers understand the causes of the disease. It can also provide patients with earlier and more accurate diagnosis.

De novo sequencing is one of the key technologies of mass spectrometry-based proteomics, especially for new proteins whose genomic information is usually limited or unavailable. Therefore, the complete de novo sequencing of unknown protein sequences is of great significance. The use of antibody de novo sequencing technology can assist researchers in carrying out antibody drug research and development related scientific research projects, but it can also greatly shorten the antibody drug development cycle and quality control of the production process, and assist in the implementation and landing of the project.

What Can Creative Biolabs Do?

Creative Biolabs de novo antibody sequencing workflow provides complete antibody sequences with unparalleled accuracy and reliability. The antibody samples were digested with multiple enzymes and the fragments were measured by LC-MS/MS to derive the de novo peptide sequence. These fragments are then analyzed through Creative Biolabs' state-of-the-art bioinformatics algorithm suite to assemble any full-length antibody sequence of any isotype. Our unique de novo sequencing workflow enables us to identify complete antibody sequences with hundreds of overlapping peptides without the need for a database or sequence library as a reference or homology. Creative Biolabs has overcome the key technical difficulties of antibody de novo sequencing, realized the accurate identification of leucine and isoleucine, and promoted the breakthrough scientific development of protein/antibody sequencing in the research, diagnosis, and treatment industries. 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. The polyvalent form of purified monoclonal antibody can cover 100% of the desired area and has excellent accuracy for sequencing. Creative Biolabs has a complete customized service chain, from antibody design and cloning to de novo sequencing and analysis, as well as in vitro optimization to meet any customer needs.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.