Introduction
Edman degradation is a time-honored N-terminal sequencing technique for proteins and cleavage fragments. Proteolytic
processing modifies the pleiotropic functions of many large, complex, and also reveal cryptic binding sites and
generate novel or modified biologically active cleavage products displaying new N- and C-termini. Proteolytic
processing is a ubiquitous and irreversible post-translational modification involving limited and highly specific
hydrolysis of peptide and isopeptide bonds of proteins by proteases. The identification of exact proteolytic
cleavage sites in the extracellular matrix laminins and other extracellular matrix proteins is not only important
for understanding protein turnover but is needed for the identification of new bioactive cleavage products. However,
the identification of cleavage sites in complex macromolecular proteins such as those that make up the ECM is not
trivial. N-terminal microsequencing of proteolytic fragments is a commonly used method to distinguish the N-terminus
of a protein from the N-terminus of its protease cleavage product will accelerate the identification of protease
substrates with broad or unknown specificity.
Fig. 1. Proteolytic processing of amyloid
precursor protein. (Sumner I E, et al., 2018)
Our Services
Over the past few decades, Creative Proteomics has focused on developing powerful protein sequencing
platforms for the characterization of proteolysates and native protein N/C termini. At Creative
Proteomics, our scientists prefer Edman degradation to determine your cleavage sites and analyze the
N-terminus of new protein fragments formed by degradation or enzymatic cleavage. It is an important tool for many
scientific areas including:
- Confirmation of the amino acid sequence for proteins, peptides and antibodies.
- Confirmation of the correct translation of a recombinant protein.
- Drug discovery for de novo sequencing of new novel proteins.
- Probe design for molecular cloning.
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.
At Creative Proteomics, our scientists developed a new liquid chromatography-mass spectrometry
(LC-MS) method to complement N-terminal Edman sequencing for N-terminal identification of protein cleavage fragments
in solution. Because N-terminal Edman sequencing of multiple and often closely spaced cleaved fragments on an
SDS-PAGE gel is difficult, limiting throughput and coverage. Our LC-MS method can identify hundreds of peptides in a
single analysis, making it ideal for identifying multiple cleavage sites in a single protein.
In addition to a clear and easy-to-understand report, you can expect:
- Expert advice directly from our experienced technicians.
- Fast turnaround with results usually within a week.
- Priority service for when results are required within 2-4 days.
- Validation study with application report available on request.
Creative Proteomics provides global customers with professional protein degradation or enzyme
digestion analysis service to analyze the N-terminus of the new protein fragment formed by degradation or enzyme
cleavage and thus to determine the cleavage site. At Creative Proteomics, our highly qualified team
works with our customers stand together on the front lines to help you solve tough research challenges. If you are
interested in our services, please contact us immediately.
References
- Rogers L D, Overall C M. (2013) Proteolytic post-translational modification of proteins: proteomic tools and
methodology. Mol Cell Proteomics. 12(12):3532-42.
- Doucet A, Overall C M. (2011) Broad coverage identification of multiple proteolytic cleavage site sequences in
complex high molecular weight proteins using quantitative proteomics as a complement to edman sequencing.
Mol Cell Proteomics. 10(5):M110.003533.
