De-novo sequencing of mAb
- If the mAb is new or theoretical sequence is unknown
- If parent hybridoma is not available
- Pieces together both variable and constant region
Do you need the full amino acid sequence of your monoclonal antibody?
De-novo sequencing of a purified mAb is valuable if the parent hybridoma cell is not available for DNA sequencing. De-novo sequencing of an antibody also works when the amino acid sequence does not match the DNA sequence.
We can help by antibody sequencing using tandem mass spectrometry. You can even use our analysis wh...
Do you need the full amino acid sequence of your monoclonal antibody?
De-novo sequencing of a purified mAb is valuable if the parent hybridoma cell is not available for DNA sequencing. De-novo sequencing of an antibody also works when the amino acid sequence does not match the DNA sequence.
We can help by antibody sequencing using tandem mass spectrometry. You can even use our analysis when there is no record of the theoretical amino acid sequence of your antibody in any database.
We perform amino acid sequencing of your antibody at the protein level using a combination of analyses. These are: Multiple protease digestion, LC MS/MS peptide analysis, and de-novo sequencing of the CDR domains by MS/MS data interpretation. The final sequencing is verified by comparison to the intact mass and N-terminal sequencing data.
What does a standard antibody de-novo sequencing service include?
Part 1: De-novo sequencing of monoclonal antibody
- First we separate the heavy and light chains.
- Then we digest with 6 different enzymes.
- Each digest (2 x 6 samples) is now analyzed by high resolution LC-MS/MS.
- Finally, we use PEAKS software to determine the peptide sequences. Following this final step we assemble the full length HC and LC.
Part 2: N-terminal Edman sequencing of heavy and light chains
- After de-novo sequencing we separate the heavy and light chains by SDS PAGE. Afterwards, we blot them onto PVDF membrane.
- Each chain is now analyzed by N-terminal Edman Sequencing.
- The analysis determines 20 to 30 N-terminal residues to get the leader sequences of the heavy and light chains.
Part 3: Intact mass determination by LC-MS
- First, we find the exact mass of the reduced heavy chain and light chain.
- In addition, we measure the exact mass of the de-glycosylated heavy chain.
- This analysis determines the precise protein mass weights of the full length HC and LC. It also confirms the homogeneity of the purified monoclonal ab.
Part 4: Verification of the assembled sequence
- For verification, we compare the assembled sequence to a database with known sequences.
- Finally, we compare the sequencing with the precise Mw’s of the HC and LC to verify the sequence.