ANTIBODY PROTEIN SEQUENCING

You need to sequence your antibody but you do not have any hybridoma cell line? Let us determine its amino-acid sequence thanks to our antibody protein sequencing service. With our state-of-the-art sequencing platform, 100µg of 90% purity sample are sufficient to provide a fully functional amino-acid sequence in 3 weeks only.

What are the advantages of antibody protein sequencing over classical antibody sequencing?

Antibody protein sequencing offers unique advantages over classical antibody sequencing from hybridoma cells:

• It makes antibody sequencing possible without any cell line. While classical sequencing is applicable to hybridomas or phage display, antibody protein de novo sequencing only requires a few micrograms of antibody protein. Thus, even when the hybridoma is lost, recovering your antibody is not an issue anymore! The sequence can be recovered directly from your antibody and can further be produced by transient or stable expression.
• It allows to characterize posttranslational modifications (and thus to verify antibody integrity and effectiveness). This can even not be seen classical DNA antibody sequencing.
• The method is adaptable to high-throughput sequencing. Thus, this approach is perfectly suitable for the sequencing of hundreds of antibodies.
• Antibody protein sequencing can be applied to all formats of antibodies without species restriction. We can sequence IgA, IgM, IgG, IgY, Fab, scFv from mice, rats, rabbits…

You are facing a situation where classical DNA sequencing is not applicable? Contact us to discuss about your project!

There are many cases where classical antibody sequencing from a hybridoma cell line is not possible. If you possess even less than 1 mg of your antibody (purity >90%), ProteoGenix can determine its exact sequence thanks to our antibody protein sequencing service. Our state-of-the-art antibody protein de novo sequencing platform combines the latest mass spectrometry technologies and the most advanced softwares for de novo protein sequencing. Our standard procedure includes:

• Enzymatic digestion of the antibody protein to obtain short overlapping peptides.
• LC-MS/MS of each peptide.
• Data processing including de novo peptide identification and automated overlapping peptide assembly.
• Delivery of a comprehensive antibody protein sequencing report.
• Optional: test expression + antibody/antigen interaction test

As an antibody production expert, ProteoGenix can back-translate the known amino-acid sequence into a gene and use it as a starting material for the transient or stable expression of your antibody. This can be part of a complete antibody development process including optimization of production. To get more information about our capabilities, contact our PhD account managers.

Leucine/Isoleucine determination

The distinction between leucine and isoleucine is especially important in the content of antibody protein sequencing. Indeed, the replacement of a leucine by an isoleucine and vice versa in the CDR region or in the vicinity of it can dramatically impact the affinity and specificity of your future recombinant antibody.
For a long time, Edman degradation was considered as the best approach for antibody sequencing and for leucine/isoleucine determination. However, modern MS techniques are more sensitive, faster (and thus cheaper) and allow characterization of longer and even modified peptides. The main problem of this technique is that it does not allow for proper isoleucine/leucine discrimination. Advanced fragmentation technologies such as electron transfer high energy collision dissociation solve this problem by generating different w-ions for leucine and isoleucine as:

• z.-ion with leucine is transformed to w-ion by isopropyl radical loss.
• z.-ion with isoleucine on its N-terminus is transformed to w-ion by ethyl or methyl radical loss.

Therefore, w-ions formed by leucine and isoleucine side chains fragmentation are characterized by different mass and become distinguishable by mass spectrometry. This technique coupled to classical digestion enzyme specificity and statistical analysis of homologous sequences in antibody database allows attributing an amino-acid residue to each position with high confidence.
Our leucine/isoleucine determination takes all these parameters into account. This results in an antibody sequence characterization with unparalleled accuracy even in the CDR region.

What is antibody protein sequencing?

Antibody protein sequencing refers to the derivation of the amino-acid sequence of an antibody without needing any access to mRNA or a hybridoma cell line. The main idea of de novo sequencing is to exploit the mass difference between two fragment ions to determine the mass of a residue in a peptide sequence. As most of the amino-acids are characterized by a unique mass (except leucine and isoleucine), it is theoretically possible to deduce the peptide sequence.
In practice, the MS/MS spectra interpretation starts with the assignment of the y and b ions. This requires the interpretation of a human expert or of a de novo sequencing software. For long, de novo antibody sequencing was considered as a time-consuming process. However, recent advances of computer algorithms such as PEAKS or NOVOR have considerably increased the performances of raw MS/MS spectra analysis even if some difficulties remain. These difficulties are of different nature:

• Presence of post-translational modifications leading to difficult mass attribution
• Presence of residues with similar mass (such as leucine and isoleucine)
• Presence of noise peaks
• Absence of fragment ions

and can lead to uncertainties in the characterization of the peptide, and thus the correct antibody sequence.
Thanks to the latest antibody protein sequencing technologies, ProteoGenix is able to overcome all these difficulties and to guarantee a sequence accuracy of at least 90%. You need to sequence your antibody? Contact our PhD account managers and let us bear your project.