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

Technical details, sample info, experts etc.

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 also works when the amino acid sequence does not match the DNA sequence.

We can help...

Do you need the full amino acid sequence of your monoclonal antibody?

We can help by de-novo sequencing of your antibody 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 sequence is verified by comparison to the intact mass and N-terminal sequencing data.

**A standard antibody de-novo sequencing service includes**

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 sequence of heavy and light chains

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 the 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.

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Testimonials

"Not only do we gain access to their hands, but we also get to pick their brains for mass spectrometry knowledge."

Protein characterization for optimization of manufacturing processes.

"With the results from the extensive characterization, we identified a scale-up problem and could optimize the process to increase mAb stability."

Combining mass spec analyses for mAb characterization and batch-to-batch comparison

Knowledge center

More information

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

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Learn more in this application note and blog posts:

Application note

Related blog posts:

Technical details

Introduction to de-novo sequencing

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 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. 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 determined by deconvolution of the obtained raw spectra using the MaxEnt algorithm. The high resolution and accuracy of the Q-TOF instrument 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 sequence information the full sequence of both the heavy and the light chains.

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 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 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 analysis by mass spectrometry requires a pure antibody in sufficient amounts to obtain good data. It is important that samples are prepared in a clean laboratory to avoid contamination with human keratin.

Antibody samples can be submitted in liquid or lyophilized form.

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, but the best result are obtained with low buffer strength in volatile solvents without detergents
3. Minimum amount ~ 0.5 mg
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

Related services

You can combine this analysis with other analyses such as:

Meet the experts

For this type of analysis our experts include:

Jakob Bunkenborg

PhD in Analytical Chemistry

Fen Yang Reske-Nielsen

PhD in Protein Chemistry

De-novo sequencing of mAb

Do you need the full amino acid sequence of your monoclonal antibody?

Do you need the full amino acid sequence of your monoclonal antibody?

**A standard antibody de-novo sequencing service includes**

The process:

Learn more:

Application note

Purity requirements

Start the collaboration

Ask a question or get a quote

Set up a phone meeting

Testimonials

"Not only do we gain access to their hands, but we also get to pick their brains for mass spectrometry knowledge."

"With the results from the extensive characterization, we identified a scale-up problem and could optimize the process to increase mAb stability."

Knowledge center

More information

Learn more in this application note and blog posts:

Introduction to de-novo sequencing

Intact mass analysis

Peptide sequencing

Edman N-terminal sequencing

Sample requirements for de-novo sequencing

Purify the antibody

Put the antibody sample into microcentrifuge tube

You can combine this analysis with other analyses such as:

Meet the experts