Protein sequencing

Protein or N-terminal sequencing

Protein Sequencing or N-terminal sequencing is the process of identifying the order of amino acid arrangements in a peptide or protein. Our service is accredited by UKAS to ISO 17025:2017.

Otherwise known as Edman degradation, this technique is recognised as the definitive protein sequencing method and is accepted as being the most reliable, robust and accurate method, providing an unambiguous sequence read.

It is an important tool for many scientific areas including:

• Identification and characterisation of proteins and peptides
• Confirmation of the amino acid sequence for proteins, peptides and antibodies
• Analysis of samples from Good Manufacturing Practice (GMP) processes
• Confirmation of the correct translation of a recombinant protein
• Drug discovery for de novo sequencing of new novel proteins
• Probe design for molecular cloning

N-terminal protein sequencing service

Our protein sequencing service is accredited to ISO/ILEC 17025:2017 and is based on the Edman degradation methodology. We can sequence samples provided on PVDF blots, as gel slices or proteins/peptides in solution or as dried material.

Our instruments are capable of high throughput and sensitive to the picomole level.
The technique is capable of reads up to 45 amino acids (sample dependent) and we can also offer positive identification of Cysteine residues for peptides or proteins supplied in solution or as dried material where required. Post-translational modifications such as hydroxyproline, norValine, norLeucine are also easily identified using our methods.

You can expect:

• Expert advice directly from our experienced technicians
• Fast turnaround with results usually within 7 days
• Priority service for when results are required within 2-4 days
• A clear and easy to understand report
• Data archived for a minimum of 7 years
• Internal standard run with every sample
• Validation study with application report available on request

N-terminal sequencing protocol

Our sequencing method, Edman degradation, is a procedure that requires a free N-terminal amino group on the peptide or protein. The cyclical process involves the coupling of sequencing reagent, phenyl isothiocyanate (PITC), to the free amino group of the N-terminal amino acid which is then selectively cleaved. This PITC coupled residue is converted to a stable PTH-residue and after separation by HPLC chromatography, the amino acid can be identified. The Edman reaction cycle is repeated for the number of cycles you require until a sequence of amino acids is determined.

If the sample is N-terminally blocked, e.g. due to pyroglutamate formation (which occurs often with N-terminal Gln(Q) residues) or acetylation then sequencing cannot be achieved. We cannot determine this prior to sequencing.

Information on preparing and sending samples for N-terminal sequencing can be found on our protein sequencing sample information page.

For more information on our protein sequencing services, please email us at info@altabioscience.com or call us on +44(0)1527 584495 to speak to one of our experts.

References

Edman P. ‘Method for the determination of the amino acid sequence in peptides.’ Acta.Chem. Scand. 4, 283-293, (1950).
4. Findlay, J. B. C. and M.J.Geisow ‘Protein Sequencing: A Practical Approach.’ Oxford University Press. New York: 1989.