Procedure of De Novo Antibody Sequencing Based on Orbitrap Fusion Lumos

Antibodies are crucial components of the immune system and play vital roles in modern biomedical research and applications. Antibody sequencing is essential for revealing the amino acid sequence of antibodies, providing critical information for vaccine development, disease diagnosis, and treatment. Recently, the de novo antibody sequencing method using the Orbitrap Fusion Lumos mass spectrometer has demonstrated significant advantages in antibody sequence analysis.

 

The de novo antibody sequencing method based on the Orbitrap Fusion Lumos mass spectrometer relies on high-resolution mass spectrometry technology. The Orbitrap Fusion Lumos is an advanced instrument known for its high resolution, sensitivity, and wide dynamic range. Antibody samples are enzymatically digested to produce peptides, which are then analyzed by mass spectrometry to determine their mass-to-charge ratios, enabling the deduction of the amino acid sequences.

 

Procedures

1. Sample Preparation

Antibody samples are first purified to eliminate impurities and non-target proteins. The purified antibodies are then digested with enzymes, such as trypsin or chymotrypsin, to generate peptides.

 

2. Mass Spectrometry Analysis

The resulting peptides are separated and detected using liquid chromatography (LC) coupled with mass spectrometry (MS). The Orbitrap Fusion Lumos provides high-resolution mass spectra, accurately measuring the peptides' mass-to-charge ratios.

 

3. Data Processing

Specialized software processes the mass spectrometry data using de novo sequencing algorithms to deduce the amino acid sequences from the mass-to-charge ratios and fragment ion information of the peptides. This approach does not rely on known antibody sequence databases, allowing direct reconstruction of antibody sequences from the mass spectrometry data.

 

Advantages

1. High Resolution and Sensitivity

The Orbitrap Fusion Lumos's high resolution and sensitivity enable accurate detection of low-abundance peptides, enhancing sequencing accuracy and comprehensiveness.

 

2. No Reference Sequence Requirement

Unlike traditional methods that depend on database searches, the de novo sequencing method can directly deduce antibody sequences from mass spectrometry data, making it suitable for novel and unknown antibodies.

 

3. Rapid and High Throughput

This method is highly automated, allowing the quick processing of numerous samples, making it ideal for large-scale antibody library screening and analysis.

 

Applications

The de novo antibody sequencing method using Orbitrap Fusion Lumos shows broad application prospects across various fields. In antibody drug development, sequencing and analyzing antibody structures help optimize antibody affinity and stability. In disease diagnosis, analyzing patient antibody sequences aids in identifying specific pathogens or disease markers. In basic research, this sequencing technology provides crucial data for studying antibody diversity and evolution.

 

The de novo antibody sequencing method based on Orbitrap Fusion Lumos, with its high resolution, sensitivity, and independence from reference sequences, has become a crucial method in antibody sequencing. As technology advances and applications deepen, this method will play an increasingly significant role in biomedical research and applications.