Is it possible to perform a de-novo sequencing of my purified monoclonal antibody without the hybridoma cells?
Yes! – Your purified antibody can be 100% sequenced at the protein level. This includes the variable CDR domains, the leader regions and the constant parts. You don’t need the hybridoma cells to get accurate sequence information!
The process starts by protease digestions. This is followed by high resolution LC MS/MS analysis to assemble the full sequence. You only need 200 ug or less of the purified mAb for the whole process.
Analysis steps for de-novo sequencing
Assemble the full sequence of the heavy and light chains of the antibody
I always start by separating the heavy and the light chains. Then I suggest each chain with a range of different proteases to obtain multiple coverage of each amino acid residue.
The peptide mixtures are analyzed by:
- Sensitive nanoLC-MS/MS.
- Database searching using PEAKS and Mascot software to analyze the MS/MS data and to search against antibody databases. What is particular great is, that the database search will give you the sequence of the constant region and identify peptides from the variable regions.
Following this analysis, the leader sequences of the heavy and the light chain are
- Obtained by standard N-terminal Edman sequencing.
- Used for assembly of de-novo peptide sequences with the assistance of the PEAKS software. The full HC and LC sequences contain the assembled leader sequence, the de-novo sequence variable parts and the constant sequence.
Confirm that the assembled sequences are overall correct by LC MS intact mass determination
- I perform this on the reduced light chain and the heavy after de-glycosylation.
Verify the assembled sequence by searching against Germline V, D, and J gene database.
- This can also help correcting the isobaric amino acids Ile and Leu in the variable regions.
“The last steps make the de-novo sequencing more reliable: First, you confirm the de-novo sequence by comparing the calculated mass of heavy and light chain. Then you compare it with other antibody sequences.”
NB: The antibody must be monoclonal with a purity above 90%, see this blog post
The antibody de-novo sequencing service that is provided by Alphalyse requires approximately 200 micrograms for a confident sequence assignment. In some cases, though, we have been able to perform successful analysis on just 100 µg.
- Download de-novo sequencing application note now and get examples of sequence coverage maps of heavy and light chain >>