Sequence Verification in De Novo Antibody Sequencing

Antibodies play a crucial role in biomedical research and therapy due to their specificity and affinity, making them ideal tools for diagnosing and treating various diseases. Accurate determination of their amino acid sequences is essential for further understanding and utilization. De Novo antibody sequencing, a technique independent of reference sequences, is gaining importance in antibody research. Sequence validation is particularly critical in this process to ensure the accuracy and reliability of sequencing results.

 

De Novo antibody sequencing directly analyzes the amino acid sequence of antibodies using mass spectrometry. This technique does not require prior knowledge of the antibody's gene sequence, making it particularly suitable for studying novel or unknown antibodies. The method involves using a mass spectrometer to fragment the antibody into peptides, measure their mass-to-charge ratio (m/z), and reconstruct the complete amino acid sequence through bioinformatics analysis.

 

Importance of Sequence Validation

Sequence validation is a crucial step in De Novo antibody sequencing. Unvalidated sequences may contain erroneous amino acid residues, mismatches, or deletions, significantly impacting subsequent functional studies and applications. Effective sequence validation ensures the accuracy of sequencing results, enhancing the credibility and reproducibility of the research.

 

Methods of Sequence Validation

1. Mass Spectrometry Validation

Repeat mass spectrometry analyses can verify the consistency of the obtained amino acid sequences. This method relies on the high reproducibility of mass spectrometry data. By comparing results from different mass spectrometry experiments, the sequence's reliability can be confirmed.

 

2. Bioinformatics Validation

Bioinformatics tools compare and analyze the sequencing results. For instance, comparing De Novo sequenced sequences with those in known antibody databases to identify similarities and differences. High sequence similarity typically indicates high reliability of the sequencing results.

 

3. Functional Validation

Experimental validation of the antibody's functionality, such as its ability to bind specific antigens, can confirm the accuracy of the antibody sequence from an application perspective. Overall functional consistency is a crucial indicator of sequence accuracy, even if some sequence variations do not affect the antibody's function.

 

4. Synthetic Validation

Synthesizing corresponding peptides or full-length antibodies based on De Novo sequencing results, followed by mass spectrometry and functional experiments for validation, is one of the most direct and reliable methods. It comprehensively confirms the accuracy of the sequencing results.

 

Sequence validation in De Novo antibody sequencing is a key step to ensure the accuracy and reliability of antibody research. Combining multiple methods such as repeat mass spectrometry analyses, bioinformatics validation, functional validation, and synthetic validation can maximize the accuracy of sequencing results. This approach not only aids in basic antibody research but also provides a solid foundation for developing antibody drugs.