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

Antibody characterization
Mar 22. 2017


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?



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


antibody de-novo sequencing

About Alphalyse

Alphalyse uses expertise in the field of protein chemistry and bioinformatics, combined with top-of-the-line mass spectrometry equipment, to provide protein analysis services globally.

Alphalyse aims to deliver high quality data and customer service to all of our clients in a fast and convenient way.

Newsletter Subscribe to The Alphalyse Newsletter and get more
information on protein analysis methods and applications
© 2019 Alphalyse, Inc.