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N- and C-terminal sequencing

Top-down sequencing of intact proteins by MALDI-ISD

Do you want to confirm the N- and C-terminal sequence of your drug substance protein?

By using MALDI-ISD we can determine up to 80 amino acids from the N-terminal of your drug substance protein. Also, we map up to 80 amino acids from the C-terminal. N- and C-terminal sequencing by MALDI-ISD works for terminally modified proteins. Such as N-terminally blocked proteins, where traditional N-terminal Edman sequencing does not work. With MALDI-ISD truncated isoforms and post translationally modified terminals can be identified.

Do you follow the ICH Q6B? (Test Procedures and Acceptance Criteria for Biotechnological or Biological Products). The ICH Q6B requires that you provide an overview of the N- and C-term amino acid sequence of your drug substance. Our experts provide you with a report to use in your project to live up to the requirements.

Our service includes:

  • N- and C-terminal sequence of your sample analysis by MALDI-ISD.
  • Determination of 20-80 amino acids from each terminal of the protein.
  • Comparison with the expected drug substance sequence.
  • MALDI-MS intact protein Mw determination.

Below you will find our simple order form. Or you can send us a request so we can discuss the analyses for your specific needs.

Download application note (pdf – 766 kb)

Services/Products
Protein analysis by Alphalyse

N- and C-terminal sequencing of drug substance with HPLC purification

  • N- & C-terminal sequence by MALDI-ISD
  • Standard UV-HPLC separation of sample
  • 1 fraction collected and analyzed
  • Comparison with DS sequence

$2,100.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote
Protein analysis by Alphalyse

N-terminal Edman sequencing of drug substance

  • N-terminal Edman sequencing
  • Determination of 10 amino acids
  • Comparison with DS sequence
  • From pure protein in-solution, lyophilized, or PVDF gel blot

$900.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote
Protein analysis by Alphalyse

Peptide mapping looking for N- and C-terminal peptides

  • Digestion with 1-3 enzymes, e.g. trypsin, Lys-C
  • nanoLC-MS/MS, 60 min gradient
  • Common modifications included, e.g. deamidation, oxidation, truncations
  • Sequence coverage generally 75-85%

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote

Technical details

Introduction
The intact protein is fragmented in the MALDI ion source. The reflector MALDI spectrum shows intense c-ions (and a-ions) for N-terminal sequencing, as well as y-ions (and z+2 ions) for C-terminal sequencing. Reflector MALDI spectra from 700-5000 Da typically cover 7-50 residues from the terminals.

Procedure
Proteins are reduced with DTT and alkylated with iodoacetamide, and subsequently purified by HPLC (C8 reverse phase column) to remove peptides and other contaminants that can obscure the analysis. The protein samples (20-50 pmol) is deposited on a stainless steel target and co-crystallized with a matrix of 2,5-DHB, 1,5-DAN, or sinapinic acid (refs 1-5). The MALDI mass spectra is acquired on an Autoflex Speed instrument (Bruker Daltronics) in ISD-mode where the protein ions spontaneously fragment to yield predominantly c-type fragments with high abundance, and y-, z- and a-ions at lower abundance. The mass spectra is calibrated using external calibration with a standard peptide calibration mixture.

The MS data is correlated to the protein sequences by Mascot database searching (Matrix Science). The Sequence Editor tool in BioTools (Bruker Daltronics) is used for de-novo sequencing and detection of sequence modifications. The database search is performed against the NCBI NRDB database containing more than 80 mill protein sequences.

Sample requirements

Sample Preparation Guidelines
Protein samples can be submitted in liquid or as lyophilized material.
The chromatographic protein purity should be >90%
Avoid detergents and keep buffer concentration at a minimum. MALDI ISD  analysis 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.
Minimum amount ~ 50-100 micrograms

Put the protein/peptide into microcentrifuge tube
Use Eppendorf Safe Lock tubes, or similar tubes from your lab
Lyophilize or speed vac the protein to a solid sample to ensure protein stability during shipment
Alternatively, freeze or refrigerate to +4°C for cold shipment of the liquid sample

Attention
Please note that MALDI ISD CANNOT be conducted on samples from gels or PVDF membranes!

Sample shipment

shipping option
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.

  • 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.
200 Page Mill Road, Suite 100
Palo Alto, CA, 94306
USA
Att: Jette Williams
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220, Odense
DENMARK
Att: Anette Rasmussen
Tel: +45 6310 6500
shipping option
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.
  • 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.

shipping option
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.
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. Fill with cooling units/cold packs.
  • 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.

shipping option
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

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