Antibodies are such potent tools that they can be utilized in many applications. Besides, antibody protein sequencing is one of the most advanced techniques that are being used in the field of medicine, genetics, and other related fields. Despite the multiple applications of antibody protein sequencing, many are still unaware of its vital roles in several science areas.

 

What is Antibody Protein Sequencing?

Simply put, antibody protein sequencing identifies the amino acid sequence of an unidentified antibody. Due to the application of mass spectrometry in the de novo sequencing of antibody proteins, it is now possible to carry out this procedure without access to the cell lines or prior knowledge of the DNA sequence.

You’ll have to consider the following benefits when comparing antibody protein sequencing services.

A More Reliable Method

 

In classical antibody sequencing, an antibody is broken into pieces by digestion with proteases. The resulting peptides are then analyzed using mass spectrometry to identify the amino acid sequence of the antibody. In a typical experiment, thousands of amino acids will be identified. However, only a fraction of these is part of the original antibody, and the rest are from other proteins in the cell lysate (the sample used for analysis). This means that some peptides may be incorrectly labeled as part of your antibody, which can lead to errors in protein identification and make it challenging to study the structure of antibodies.

Antibody protein sequencing avoids this problem because it uses intact antibodies rather than fragments. All peptides identified come from your antibody, so there’s no possibility of labeling errors (except when you’re sequencing an antibody fragment). It also reduces the complexity of analyzing large numbers of peptides at once and reduces cost since fewer experiments must be performed per sample.

More Accurate Than Classical Antibody Sequencing

Antibody protein sequencing is more accurate than classical antibody sequencing because it goes through many steps before it even gets to your computer screen for analysis. First, the sample must be prepared so that there are no contaminants or other proteins in your sample that could interfere with your results. Also, Classical DNA and RNA sequencing detects all parts of an antigen, including non-specific parts (regions with low homology). This could lead to false results compared with other methods, such as ELISA or western blotting.

Rapid Sequencing

The reason is that it uses an additional step in identifying antibodies. Classical antibody sequencing requires RNA amplification followed by reverse transcription and PCR amplification. Antibody protein sequencing only requires RNA amplification, and next-generation sequencing (NGS) reads from the variable regions of the heavy chain and light chain genes.

A Much Cheaper Alternative

 

This is because no reagents are needed for antibody protein sequencing other than those used for NGS reads from the variable regions of the heavy chain and light chain genes. This means that there are no costs associated with reverse transcription or PCR amplification, which are expensive processes that require reagents such as dNTPs (deoxynucleoside triphosphates). It will also be easier to use than classical antibody sequencing because it does not require special equipment or skills in handling biohazard materials such as viral cultures and cell lines, making it a safer method for handling pathogens.

5. Provides More In-depth Analysis

Protein sequencing provides a more in-depth antibody analysis, including the sequence of the light and heavy chains. It can also be used to determine if there is any post-translational modification (PTM) of the antibody. Post-translational modifications are covalent modifications made by enzymes or other protein molecules after the protein has been translated from messenger RNA.

Protein sequencing is also helpful in determining where an antibody has been expressed and identifying any potential antibodies with similar sequences that may have been produced by other cells.

This information found during protein sequencing helps understand how a particular antibody works within a cell and its interaction with other proteins to accomplish its function.

It’s important to note that protein sequencing is not as sensitive as classical antibody sequencing because it does not look at every single amino acid in the protein structure. However, it provides valuable information about how an antibody functions within its target cell population.

#Bonus: Helps Identifies Glycosylation And Disulfides

 

With antibody protein sequencing, investigators can identify the different glycosylation and disulfides that occur in an antibody. This is a significant advantage over classical antibody sequencing, which can only determine the type of antibody and its class.

The two types of modifications that occur on antibodies are essential in identifying differences between different strains or species. Disulfides can change where a heavy chain binds with a light chain, which affects antigen binding properties. In contrast, glycosylation can affect how long an antibody will stay in circulation before it is cleared from the body.

Meanwhile, classical antibody sequencing works by comparing amino acid sequences with those of known antibodies. This allows researchers to determine what type of antibody they have isolated from their sample — a monoclonal or polyclonal antibody — and whether it has been modified by glycosylation or disulfide bonds.

Conclusion

Looking at the world of Antibody protein sequencing, we can be sure that the future will be exciting, and we can expect our quality of life to begin to change dramatically. Several studies have been conducted concerning antibody protein sequencing. Understanding the advantages of antibody protein sequencing is vital for its successful application.