一．Why do We Need to Perform Full-length Antibody Sequencing?

There are two general methods for recovering the heavy and light chain sequences of monoclonal antibodies. The most direct method is to sequence the transcripts or genetic material of B cells. This method is applicable to hybridomas, phage display, yeast display or single-cell screening, and is the most cost-effective and reliable method for antibody sequencing. However, the source cells are not always available. If the hybridoma is lost, the genetic material may not be available. In this case, antibody sequencing can be used to recover the antibody sequence, such as using Edman degradation or mass spectrometry.

二．Classification of Antibody Sequencing

1. Sequencing based on Edman degradation: It is a mature protein sequence determination technology that starts from the N-terminus and reads one amino acid at a time. The main advantage of Edman degradation sequencing is the low sample volume requirement (usually less than 1ug is required). The quality of Edman degradation sequencing decreases with the increase in the number of amino acid processing, so this sequencing method is usually used to determine the first 30-50 amino acids and is rarely used for antibody full sequence determination.

2. Mass spectrometry-based antibody sequencing: The antibody is broken down into 15-20 amino acid long peptides, which are then analyzed in a mass spectrometer. Ideally, each peptide produces a fragment map. When performing de novo antibody sequencing, each fragment map needs to be parsed and a portion of the antibody sequence is revealed. Typically, we use multiple enzymes with different cleavage motifs to break down antibodies, ensuring that each region of the antibody can produce several overlapping peptides. By producing overlapping peptides, antibody sequencing technology can reassemble antibody sequences in a manner similar to shotgun sequencing to assemble genomes.

3. Peptide mapping and de novo antibody sequencing: The goal of peptide mapping is to confirm known sequences, and de novo antibody sequencing is the process of obtaining previously unknown sequences from fragment maps or collections of maps. Compared with de novo antibody sequencing, peptide mapping often requires fewer enzyme digestions and mass spectrometry runs, which means that costs can be lower.

三．Protein Denovo Sequencing Technology

Protein de novo sequencing technology (De Novo Sequencing) is an analytical process that uses tandem mass spectrometry (MS/MS) to obtain ions, calculate the mass of amino acid residues on the peptide chain based on the mass difference between two fragment ions, and thus obtain the amino acid sequence of the protein. This technology mainly targets the genome sequence of unknown species, constructs different types of genomic DNA libraries, and performs sequence determination. Then, the sequences obtained by sequencing are spliced, assembled, and annotated by bioinformatics methods to obtain a complete genome sequence map of the species. Using whole genome de novo sequencing technology, the whole genome sequence of animals, plants, bacteria, and fungi can be obtained, thereby promoting the research of the species.

四．Experimental Process of De novo Sequencing Technology

First, the sample is subjected to a multi-enzyme hydrolysis experiment, and the peptides after enzymatic hydrolysis are subjected to mass spectrometry detection. The mass spectrum after data collection is analyzed using a de novo algorithm to obtain the corresponding peptide sequence. After alignment and splicing between peptides, it is assembled from the N-terminus to the C-terminus of the protein to obtain the amino acid sequence of the protein.

At present, liquid chromatography can be combined with Obitrap Fusion high-resolution mass spectrometer to establish a protein de novo sequencing platform, which ensures the sensitivity of identifying low-abundance peptide fragments. At the same time, the fragmentation mode of HCD combined with ETD is adopted in the peptide fragmentation process to distinguish the isomers of leucine/isoleucine.

五．Data Analysis

(1) Peptide coverage Based on specific and non-specific proteases (Trypsin, Chymotrypsin, Glu-C, Lys-C, Pepsin, Elastase, etc.), protein samples are digested separately to achieve 100% protein coverage and produce abundant and highly overlapping peptide types, and finally obtain high-quality data required for sequencing.

(2) Peptide splicing HCD will generate a series of b/y ions, and the sequence of the peptide is deduced based on these ion information. However, since multiple enzyme digestions will produce many repeated peptides, a series of peptide screening is required. First, the high-peak peptides should be selected, which is actually to judge the fragmentation of the peptide secondary ions. However, when fragment ions are missing, database searches may still be successful. Therefore, in order to improve the accuracy of de novo sequencing, the method used may combine different ion fragmentation technologies to produce multiple spectra.

(3) Distinguishing between leucine and isoleucine In the past, Leu and Ile were generally considered indistinguishable by MS because their molecular weights were exactly the same. Incorrectly positioned Leu/Ile residues in the variable domain, especially in the CDR (complementarity determining region) of antibodies, may lead to a substantial loss of antigen binding affinity and antibody specificity.

(4) Leucine and isoleucine can obtain c/z ions under ETD fragmentation conditions, and then assist HCD fragmentation to remove a part of the group on the basis of the z ion to form w ions. Leucine removes the propyl group, resulting in a loss of 43Da; isoleucine removes the ethyl group, resulting in a loss of 29Da.

(5) Data verification: Molecular weight verification The basic modification information of the protein (oxidation, deamidation, N-terminal cyclization, C-terminal K loss, glycosylation modification) can be obtained through the protein molecular weight (intact, reduced, deglycosylated, deglycosylated reduced), thereby verifying the protein sequence obtained by De Novo. A new database was established for the sequence obtained by De Novo determination, and the coverage analysis of the collected mass spectrometry data was performed again to obtain an almost complete match result.

Tek Biotech has been committed to protein expression and sequencing research for many years. Protein amino acid sequence analysis is the basis and key to protein research. The information obtained through protein sequencing has many valuable uses, such as protein identification, probe design for molecular cloning, and peptide synthesis for immunogens. Based on high-resolution mass spectrometry and high-fidelity gene cloning technology, we can provide customers with high-fidelity antibody and protein sequencing services.