CAR-T, therapeutic antibody, bispecific, T-cell engager, or other biologics research relies heavily on sequencing. Different sequencing methods exist, and it is important to weigh cost and bandwidth when deciding when in the discovery process to obtain sequences.

Antibody sequencing can help you acquire your antibody sequences quickly, accurately, and with few barriers to entry, whether you have a single lead clone or a panel of antibody candidates. Full-length, high-confidence reads of the entire antibody variable region are attainable using powerful molecular biology techniques and Sanger sequencing methods.

The following part walks through the standard sequencing techniques.

1. Sanger Sequencing Technique

Sanger sequencing, a low-throughput technique, was one of the first to determine the antibody nucleic acid sequence. For Sanger sequencing, a DNA sample is divided into four tubes containing all four deoxynucleotides and DNA polymerase.

As the DNA in each vial is transcribed, adding modified labeled di-deoxynucleoside triphosphates (ddNTPs) stops the transcription process. A unique ddNTP is used for each sample to halt the sequencing process at a distinct nucleotide. The electrophoretic separation of the resulting fragments can then be used to determine the DNA sequence by comparing the mobility of the various bands.

This technique was used to sequence the first complete human genome as part of the Human Genome Project in 2003. It has since found widespread application across various fields, including antibody research. However, this approach has its limitations as it is slow, laborious, and prone to mistakes. However, numerous life science experts offer alternative Antibody Sequencing Services designed to increase sequencing output while maintaining high accuracy.

2. De Novo Peptide Sequencing

The Latin phrase “de novo” means “from scratch” or “all over again.” De novo peptide sequencing is the process of determining a peptide’s amino acid sequence from scratch. Peptide mass fingerprinting and other database-dependent methods have limitations that can be avoided by using this technique to obtain the peptide sequences (PMF). It employs computational methods to infer the peptide sequence from MS/MS spectra acquired in experiments. This technique is applicable for endogenous peptides, peptides with posttranslational modifications (PTMs), antibodies, and unsequenced organisms.

3. Edman Sequencing

Edman degradation, created by Pehr Edman, is a method for determining the amino acid sequence of a protein or peptide. The peptide bonds between other amino acid residues are preserved while the N-terminus is labeled and cleaved off using this method. In 1967, Edman and Beggs developed an automated process for the Edman degradation reaction. Nowadays, peptides up to 50 amino acids in length can be sequenced using the automated Edman degradation (protein sequenator).

Edman degradation is a three-step process that involves coupling phenylisothiocyanate (PITC) to the -amino group of a peptide or protein. Further, it consists in cleaving the amino-terminal amino acid (via cyclization in strong per-fluorinated acid, typically trifluoroacetic acid (TFA), to a 2-anilino-5-thiazolinone) and then converting the resulting thiazolinone.

The optimal pH for coupling is above the pK of the amino group (pH=7.8), where it is uncharged, and below the pH of 10, where PITC is hydrolyzed. The peptide and the PITC reagent’s solubility are improved by the coupling medium, a combination of aqueous and organic solvents.

4. Mass Spectrometry

The mass-to-charge ratio of ions allows mass spectrometry (MS) analysis of proteins to separate and quantify individual molecules from complex mixtures. In recent years, MS has proven its worth in a variety of different settings, including proteomics. Over the past two decades, advances in high-throughput and quantitative MS proteomics workflows have significantly increased our understanding of protein structure, function, modification, and global dynamics.

The mass-to-charge (m/z) ratio of molecules allows mass spectrometry to be used as a sensitive tool for detecting, identifying, and quantifying them. First used in the life sciences to track heavy isotopes, MS was developed nearly a century ago to measure the atomic weights of elements and the abundance of specific isotopes. Later, MS was utilized for peptide and oligonucleotide sequencing and nucleotide structure analysis.

Improved macromolecule ionization techniques like electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) made MS a viable tool for studying protein structure. With the aid of ionization, researchers were able to obtain protein mass “fingerprints” that could be compared to known proteins and peptides in databases.

The relative and absolute amounts of target proteins were measured thanks to advances in isotopic tagging techniques. Thanks to these developments in technology, techniques have been developed for effectively analyzing samples in the solid, liquid, or gaseous states.

5.  Single-Molecule Protein Sequencing

While high-throughput DNA-sequencing technologies can probe individual molecules, protein sequencing typically employs ensemble techniques and necessitates more of a pure starting material. Proteins are crucial components of living organisms. Hence, knowing how much protein a cell or organism contains can shed light on various physiological processes and diseases. Despite the value of protein analysis, only a few methods exist for deciphering protein sequences, and these have drawbacks, such as sample size requirements. Proteomics research would be entirely transformed by the advent of single-molecule techniques, allowing for the most sensitive detection of low-abundance proteins and the actualization of single-cell proteomics.

Final Thought

Regardless of your target’s nature, antibody sequencing techniques provide the rapid, dependable, and high-quality method you need to take your discovery to the next level. Whether you need the sequence, a purified antibody made through recombinant expression, or a molecular model of the CDRs, these techniques are suitable for the sequencing of hundreds of antibodies.