Verifying Ile/Leu residues in de-novo sequencing of antibody

Antibody characterization
Alphalyse
Mar 22. 2017

Question:

I was told that de-novo sequencing by mass spectrometry is not able to distinguish between Ile and Leu residues in my antibody.
How can I verify Ile & Leu residues when doing de-novo sequencing?

 

Answer:

A typical problem with LC MS/MS peptide sequencing relates to the isobaric amino acids, i.e. amino acid residues of identical mass. The issue arises because the sequence obtained by mass spec cannot distinguish between Leucine and Isoleucine, and in a few cases between glutamine and Lysine.

“To avoid the problem, I suggest you evaluate the I/L assignments. Consider the enzymatic cleavage specificity, and also compare with the germ line V, D, and J gene sequences for antibodies.”

Another possible solution is to collect selected peptides by HPLC. Then you can follow up with traditional peptide sequencing by Edman degradation. This will establish the correct sequence and I/L verification and other isobaric amino acid combinations.

 

Get help with Antibody analysis

If you don’t feel confident in performing these analyses yourself, have a look at the Alphalyse Antibody de-novo webpages. There’s a request form, which you can fill in for more information.

Visit Alphalyse for information on how to order this analysis.

You can also ask for an antibody de-novo analysis sample report to see what typical results look like:

Request a sample report

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antibody de-novo sequencing

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