Do you need the amino acid sequence of your new monoclonal antibody?
We can help by de-novo sequencing of your novel antibody using tandem mass spectrometry. We can even perform this analysis if the theoretical amino acid sequence is not recorded in any database.
De-novo sequencing of a purified mAb is valuable if the parent hybridoma cell is not available for DNA sequencing. You can also use de-novo sequencing when the amino acid sequence does not match the DNA sequence. We perform amino acid sequencing of your antibody at the protein level using a combination of the following analysis; multiple protease digestion, LC MS/MS peptide analysis, searching antibody sequence databases to retrieve the constant Fc regions, and de-novo sequencing of the CDR domains by MS/MS data interpretation.
Our antibody sequencing service includes:
Our experts use several techniques to piece together the variable and constant sequence. They also verify the assembled sequence:
Part 1: De-novo sequencing of monoclonal antibody using PEAKS software.
- 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 the heavy and light chain.
- First we separate the heavy and light chains by SDS PAGE. Afterwards, we blot them onto PVDF membrane.
- Each chain is now analyzed by 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 Mass weights of the full length HC and LC. It also confirms the homogeneity of the purified monoclonal ab.
Part 4: Verification of assembled sequence.
- For verification, we compare the assembled sequence to a database with known antibody sequences.
- Finally, we compare the sequence with the precise Mw’s of the HC and LC to verify the sequence.
Optional Part 5: N-terminal Edman sequencing of selected peptides in mAb.
- We can separate the protein digest by UV-LC MS/MS, and collect peptide fractions.
- N-terminal Edman sequencing can then be performed on selected peptides. We do this to elucidate and verify ambiguous residues, and to distinguish Ile and Leu.