Overview of Antibody De Novo Sequencing

Classification of Antibody De Novo Sequencing

Antibody de novo sequencing is categorized based on the type of antibody samples. We typically describe antibody samples by their "clonality," which refers to the number of different antibody sequences or genetic lineages present in the sample.

 

1.Monoclonal

The sample contains antibodies with identical sequences. Monoclonality is essential for antibody de novo sequencing. In rare cases, simple mixtures of monoclonal antibodies can undergo proteomics sequencing, but success is not guaranteed.

 

2. Polyclonal

The sample contains antibodies with diverse sequences, usually within a broad range. Antibodies derived from serum are characterized as polyclonal antibodies.

 

3. Oligoclonal

The sample contains a limited number of antibody lineages, defined by complementarity determining regions (CDRs).

 

Based on these classifications, antibody de novo sequencing is divided into monoclonal antibody de novo sequencing, polyclonal antibody de novo sequencing, and oligoclonal antibody de novo sequencing. This article primarily discusses monoclonal antibody de novo sequencing.

 

Why Do We Need Antibody De Novo Sequencing?

There are two general methods for reconstructing the heavy and light chain sequences of monoclonal antibodies. The most direct approach is sequencing the transcripts or genetic material of B cells. This method is suitable for hybridomas, phage display, yeast display, or single-cell screening, making it the most cost-effective and reliable method in antibody sequencing. However, source cells may not always be available. If a hybridoma is lost, the genetic material might be unobtainable. In such cases, antibody sequencing can be used to reconstruct the antibody sequence, using techniques such as Edman degradation or mass spectrometry.

 

Sequencing Based on Edman Degradation

Edman degradation, also known as Edman sequencing, is a well-established protein sequencing technique that reads one amino acid at a time from the N-terminus. The main advantage of Edman degradation sequencing is the low sample requirement (typically less than 1 µg). The quality of Edman degradation sequencing decreases as the number of processed amino acids increases, so this method is usually used to determine the first 30-50 amino acids and is rarely used for full antibody sequencing.

 

Mass Spectrometry-Based Antibody Sequencing

Mass spectrometry has become the preferred method for full antibody sequencing. In almost all mass spectrometry protocols used for antibody sequencing, the antibody is first digested into peptides 15-20 amino acids long, which are then analyzed by the mass spectrometer. Ideally, each peptide produces a fragmentation spectrum. During antibody de novo sequencing, each fragmentation spectrum is analyzed to reveal part of the antibody sequence. Usually, multiple enzymes with different cleavage motifs are used to digest the antibody to ensure each region generates several overlapping peptides.

 

Peptide Mapping and Antibody De Novo Sequencing

The goal of peptide mapping is to confirm a known sequence, while antibody de novo sequencing is the process of obtaining previously unknown sequences from fragmentation spectra. Compared to antibody de novo sequencing, peptide mapping often requires fewer digestions and mass spectrometry runs, resulting in lower costs.

 

Antibody de novo sequencing process: First, the antibody sample is digested with multiple enzymes. Each enzyme has a different cleavage pattern (indicated by different colors), and none of the enzymes generate new peptides from the antibody sequence. Antibody de novo sequencing uses overlapping peptides produced from multiple enzyme digestions to achieve complete antibody coverage, ensuring that no sequences are missed.

 

Advantages of Monoclonal Antibody Sequencing

What are the advantages of monoclonal antibody sequencing? Here is a summary based on MtoZ Biolabs' monoclonal antibody de novo sequencing technology:

1. Achieves 100% full sequence assembly of the antibody's heavy and light chains.

2. Combines mass spectrometry analysis software such as PEAKS with targeted data processing algorithms, ensuring no valuable data is missed.

3. Capable of sequencing various subtypes and different types of antibodies.

4. Implements rigorous sequence prediction and strict sequence verification mechanisms, providing only fully matched results.