Introduction
The N-terminal sequence composition has a great influence on the overall biological function of biopharmaceuticals
(proteins, antibodies, and vaccines). N-terminal sequencing analysis of biopharmaceuticals helps to identify their
higher-order structures while interpreting them to reveal their biological functions. For protein drugs, N-terminal
sequencing analyze protein modification sites, such as the N-terminus of proteins and peptide drugs, analyze the
position of artificial modifications such as cyclization modification and methylation modification to improve its
degradation stability and prolong drug efficacy. In addition, multiple subunits, such as reduced monoclonal
antibodies, can also be sequenced after separation by SDS-PAGE. Edman degradation N-terminal sequencing analysis is
widely used as a simple biological product identification test, complements the confirmation of impurity sequence by
mass spectrometry, and can simplify the identification of leucine-isoleucine, which is difficult for mass
spectrometry.
Fig. 1. Automated Edman peptide
sequencing. (Gauthier J, et al., 2019)
Our Services
The analysis and confirmation of the N-terminal sequence of protein and polypeptide drug molecules is an important
link in the quality control of the pharmaceutical industry. In particular, the ICH Q6B guideline requires the
N-sequence information of protein (peptide) drugs. Creative Proteomics, as the world's leading
provider of protein sequencing services, provides you with high-quality one-stop biopharmaceutical N-terminal
sequencing services with first-class experimental platforms and mature technical means.
Our Edman degraded N-terminal sequencing technology can provide N-terminal sequencing analysis methods for
biopharmaceutical proteins or other biological reagents, complementing mass spectrometry sequencing to obtain
comprehensive N-terminal sequence analysis information.
Service Process
To decode the N-terminal sequence of biopharmaceuticals, Creative Proteomics' scientists use the
following N-terminal sequencing protocol:
- Using SDS-PAGE to separate multiple proteins or protein mixtures, and then bloting onto PVDF membrane and stain.
(Pure or single-chain proteins can be transferred directly to PVDF membranes.)
- Blot samples were then treated with phenylisothiocyanate.
- The derivatized N-terminal amino acid is then cleaved from the protein backbone.
- Derivatized amino acids are analyzed by liquid chromatography and identified based on the elution position of
the derivatized amino acid standard.
What Can We Provide?
- Identify The Higher Order Structure of Proteins
- N-Terminal Sequence Analysis
- N-Terminal Modification Site Analysis
- Sequencing Light and Heavy Chain Terminals
Sample Requirments
- Sample state: Dry powder and liquid samples are accepted. (The liquid samples cannot contain protease.)
- Sample concentration and purity: Total amount > 200 μg, purity > 90%, concentration > 0.5 mg/mL.
- The recommended total amount is 2-3 mg if you need to test a large number of amino acids.
- Salt content: Volatile inorganic salts < 20 mM, non-volatile inorganic salts < 5mM.
- It is recommended to provide the theoretical sequence of the sample.
- If there is no signal peak in the sample, it is recommended to block the N-terminal of the protein and use mass
spectrometry for sequencing.
Applications of Biopharmaceutical N-Terminal Sequencing
N-terminal sequence analysis has many different applications in the development of biopharmaceuticals, including but
not limited to.
- Indicate that the N-terminus of your protein is intact and as expected.
- Demonstrate batch-to-batch consistency.
- Clearly define isoleucine and leucine residues in protein sequences.
- Confirm the sequence of the protein.
Creative Proteomics is a reliable biopharmaceutical partner. Our professional team can provide
customers with comprehensive biopharmaceutical protein N-terminal sequence information, and realizing the analysis
of protein advanced structure and modification sites. We will provide you with detailed experimental procedures and
data reports such as the final N-terminal sequence information analysis report. Please contact us immediately If you are interested in our services.
References
- Gauthier J, Vincent A T, Charette S J, et al. (2019) A brief history of bioinformatics. Brief
Bioinform. 20(6):1981-1996.
