De-novo sequencing of mAb

Full de-novo sequencing of monoclonal antibodies

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. You can even use our analysis when there is no record of the theoretical amino acid sequence of your antibody in any database.

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 perform amino acid sequencing of your antibody at the protein level using a combination of analysis. These are: 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 de novo sequencing service includes:

Our experts use several techniques to piece together the variable and also the constant sequence. In addition, they 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 sequence 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.
  • Afterwards, N-terminal Edman sequencing is performed on selected peptides. We do this to elucidate and verify ambiguous residues, and to distinguish Ile and Leu.
Protein sequencing

De-novo sequencing of antibody

  • Intact mass of reduced and deglycosylated mAb
  • Separation of HC and LC, Edman sequencing – 20-30 residues
  • Six protease digests and high resolution MS/MS
  • PEAKS de-novo sequence analysis

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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 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 multiply protonated molecules with a distribution of ion species at m/z range from 1-3000. 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.

Peptide sequencing

First we reduce the antibody then 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 to 2000 dalton 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.

Edman N-terminal sequencing

Based on MS peptide data and De-Novo sequencing data, we now select 5-10 peptides for N-terminal Edman sequencing to verify/identify the exact amino acid sequence of the variable regions. Peptide 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 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.

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.

  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, urea or detergent, but the best result are obtained 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 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

Ship your protein samples by a courier company of your own choice

Place your samples into micro tubes and pack in a suitable transport box. Remember to print and include the sample submission form.

  • Contact a courier company of your choice (FedEx, UPS, DHL, TNT, World Courier etc.). Use the following sentence to describe the content: “Protein material – Non-toxic, non-hazardous, for research purpose only“.
  • Use cool packs or cooling units if your samples should be stored below 5°C/40°F
  • Ship the samples to the office closest to you (Canadian customers, please ship to Europe):
Customers in the USA
Alphalyse, Inc.
299 California Avenue, Ste 200
Palo Alto, CA 94306
Att: Thomas Kofoed
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220 Odense SO
Att: Anette Rasmussen
Tel: +45 6310 6500
Order a prepaid FedEx Air Waybill < 1 lb./ 500g

A prepaid FedEx Air Waybill can be used for fast and easy shipment of samples in packages up to 1 lb./ 500g.
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack in suitable transport box. Remember to print and include the sample submission form.
  • Print the PDF document and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to send your samples stored at room temperature, this option is usually the right choice. If you fill your box with cooling packs, the weight will most often exceed 1 lb./500g.

Order a prepaid FedEx Air Waybill < 10 lb./5kg (e.g. for packages with cooling packs)

This prepaid FedEx Air Waybill can be used for shipment of samples in packages up to 10 lb./ 5kg, with or without cooling packs (do not use dry ice!).
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack them in a suitable transport box. Remember to print and include the sample submission form.
  • Fill with cooling units/cold packs (do not use dry ice!).
  • Print the PDF and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to fill your transportation box with cooling packs, select this shipping option. If you plan to use dry ice, you cannot use this Waybill!

Get a quote for shipment by World Courier

The World Courier service can be used for shipment of samples of all types. If you select this option, we will contact you to provide a quotation based on location and shipping conditions. You will receive a prepaid Air Waybill which will be invoiced together with your analysis results. This is usually a good option if FedEx does not service your location.
With the prepaid World Courier service you get:

  • GxP compliant processes to ensure maximal security and traceability
  • Door-to-door solution on a dedicated vehicle
  • Possibility of providing qualified packaging, temperature monitoring and dry ice
  • Shortest possible transit-time through “next flight out”-procedure
  • Customs clearance and help with paperwork
Order analysis now
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