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. It is also used when the amino acid sequence does not match the DNA sequence. We determine the amino acid sequence 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, and to 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, the peptide sequences are determined by analysis in the PEAKS software. Following this final step the full length HC and LC are assembled.
Part 2: N-terminal Edman sequencing of the heavy and light chain.
- The heavy and light chains are separated by SDS PAGE and then blotted 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. This is done to elucidate and verify ambiguous residues, and to distinguish Ile and Leu.