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 molecular 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.

Please send us a request for de-novo sequncing or have a look at our full range of antibody characterization services.

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The process:

  • 1 Contact us to discuss your project and receive a project proposal
  • 2 Analysis phase, where a project leader is appointed to manage the project
  • 3 You receive a report with a clear presentation of the findings
  • 4 Follow-up by phone or email

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    More information

    We would like to help you as much as possible with your project and therefore provide several kinds of customer support:

    Technical details

    Introduction to antibody sequencing by de-novo

    Mass spectrometric de-novo sequencing consists of two or three steps. 1) Intact mass determination, 2) peptide mapping using de-novo sequencing software and 3) Edman N-terminal sequencing of peptides.

    Intact mass analysis

    By ESI-MS, we spray the antibody sample in liquid phase through a capillary at high voltage into the MS instrument. Here, the mass over charge ratio (m/z) is then measured. For antibodies the electrospray process results in multiple protonated molecules with a distribution of ion species at m/z range from 1 to 3000. We determine the mass of the antibody with a deconvolution algorithm that calculates the mass of the intact non-protonated antibody. Note that the ESI process requires that the sample preparation is quite pure without interfering salts or detergents.

    We can also purify the sample by reversed phase HPLC (Agilent 1200 system) using a short C8 column before being analyzed on a Q-TOF mass spectrometer (Bruker Maxis Impact system). The mass of the sample is then determined by deconvolution of the obtained raw spectra using the MaxEnt algorithm. The high resolution and accuracy of the Q-TOF instrument usually result in very accurate mass determination within 0-3 Da of the theoretical mass.

    Molecular Weight Determination for de-novo sequencing

    Intact mass analysis of antibody by ESI-MS

    Peptide sequencing

    First, we reduce the antibody and then we separate the heavy chain and the light chain by HPLC. Next, we cleave each fraction into smaller peptides using 5 different, specific proteases. The peptides are analyzed by mass spectrometry and all the observed peptides having mass/charge ratio within 300-2000 Da are selected for MS/MS sequencing.

    The combined data sets are analyzed using PEAKS de-novo sequencing software collecting without any sequencing information the full sequence of both the heavy and the light chains.

    de-novo sequencing of antibody

    PEAKS De-Novo sequencing results after digest and LC-MS/MS analysis

    Edman N-terminal sequencing

    Based on MS peptide data and de-novo sequencing data, we select 5-10 peptides for N-terminal Edman sequencing. We do this to verify/identify the exact amino acid sequence of the variable regions. Up to 25 amino acid residues can be sequenced.

    The Edman degradation chemistry is a cyclic procedure where the PITC reagent couples to the free N-terminal amino group and cleaves off one amino acid at a time from the protein. We then transfer the PITC coupled residue to a flask, convert it to a PTH-residue and identify the amino acid by HPLC chromatography. The Edman degradation reaction cycle repeats to determine a sequence of N-terminal amino acids.

    Sample preparation

    Sample requirements for de-novo sequencing

    Antibody sequencing by mass spectrometry requires a pure antibody in sufficient amounts to obtain good data. It is important to prepare the sample in a clean laboratory to avoid contamination with human keratin.

    Submit antibody samples in liquid or lyophilized form.

    1. Purify the antibody

      1. The chromatographic antibody purity should be >90%.
      2. Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS can be done on samples containing small amounts of salts and urea. However, we obtain the best result with low buffer strength in volatile solvents without detergents.
      3. Minimum amount ~ 0.5 mg.
    2. Put the antibody sample into microcentrifuge tube

      1. Use a Eppendorf Safe Lock tube supplied, or similar high-quality tubes from your lab.
      2. Lyophilize or speed vac the antibody to a solid sample to ensure stability during shipment.
      3. Alternatively, freeze or refrigerate to +4 oC for cold shipment of the liquid sample.

    Now you are ready to send you sample off for de-novo sequencing.

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    Meet the experts

    For this type of analysis our experts include:

    Do you need help?
    Stine Thyssen

    PhD in Health Science

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    Do you need help?
    Li Peng Lundgren

    PhD in Protein Chemistry

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