Understanding the structure and sequence of proteins is crucial for the characterisation of protein drugs. C-terminal sequencing, which enables scientists to determine the precise amino acid composition at the carboxy-terminus of a protein, is an essential component of protein analysis.
Creative Proteomics offers C-terminal sequencing services that are compliant with section 6.1.1 c of the ICH Q6B recommendations.
What is C-Terminal Sequencing?
C-terminal sequencing, a valuable tool in molecular biology, is the process of deducing the sequential arrangement of amino acids at the C-terminus of a protein. The C-terminus is characterized by its terminal carboxyl group (-COOH), demarcating the end of a protein's polypeptide chain. Thoroughly decoding the C-terminus sequence contributes significantly to our understanding of the protein's primary structure. Further, it paves the way for a detailed assessment of its biological functionality - a critical aspect of studying protein behavior in depth.
Fig 1. Determination of C-terminal amino acid residues by use of hydrazine. (BHAGAVAN, N. V., 2002)
Typically, this sequencing process is executed through advanced methodologies such as Edman degradation or mass spectrometry-based procedures. Both these techniques harness a consistent, stepwise removal of individual amino acids from the C-terminus of the protein. Subsequent identification and quantification of these removed amino acids play a vital role in attaining an accurate sequence structure at the C-terminus.
C-Terminal Sequencing Procedures
The method for sequencing the C-terminal of a protein is a meticulous, multi-step process that encompasses both the isolation and further analysis of the protein's structure. The steps involved in this method are as follows:
Isolation and purification of the protein
The initial procedure involved requires the isolation and purification of the specific protein through a variety of methods like filtration, centrifugation or chromatography.
Carboxypeptidase digestion
Following purification, the procedure of carboxypeptidase digestion is undertaken. This involves applying the carboxypeptidase enzyme to the now purified protein solution, which sequentially cleaves off the peptide bonds from the C-terminal end, ultimately releasing individual amino acids.
Identification of released amino acids
To identify the released amino acids, techniques such as thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC) are employed.
Determination of sequence
Following their isolation, the sequencing of these amino acids is executed through techniques including Edman degradation, mass spectrometry, or automated sequencers, depending on the specific circumstances of the protein.
Repeating the process
The process above is repeated in the event that the C-terminal of the protein is not fully sequenced after the initial round. The purification and carboxypeptidase digestion steps would be carried through until the full C-terminal amino acid sequence is discerned.
Analysis and interpretation
Lastly, the sequence acquired is strategically analyzed and interpreted. This critical step aids in comprehending the protein's structure, function, and overall properties.
Fig 2. Schematic representation of the different steps in the C-terminal sequencing method (Samyn, B., et al.; 2006)
What We Provide?
Our team of experienced scientists utilizes advanced bioinformatics tools and databases to ensure high-quality analysis and interpretation of C-terminal sequencing data.
C-Terminal Sequencing Analysis
The investigation of proteins' C-terminal regions is our main area of expertise. To pinpoint the precise arrangement of amino acids at the C-terminus, we use cutting-edge methods like mass spectrometry, Edman degradation, and sophisticated bioinformatics.
N-terminal vs. C-Terminal Sequencing
We provide a contrast of N-terminal and C-terminal sequencing. This makes it possible to comprehend protein structure and function in detail, giving important insights into the overall structure of proteins.
PTM Characterization
We offer a C-Terminal Sequencing service that can locate and describe different post-translational modifications (PTMs) that are located there. Understanding protein regulation and functionality depends on accurate PTM identification.
Protein Engineering and Design
We are able to help with protein engineering and design because to our expertise in C-Terminal sequencing. We can design proteins with functional features, enhance protein expression, or change protein stability by precisely defining the C-terminal amino acid sequence.
Proteomic Analysis
We offer proteome analysis services to simultaneously examine the C-terminal sequences of many proteins in addition to individual protein analysis. This makes it possible for researchers to comprehend the overall structure and function of proteins within a biological system
Customized Service
We provide specialized data interpretation and analysis, delivering comprehensive reports with annotated sequences, discovered PTMs, structural insights, and functional implications. Our reports are created to specifically address the requirements of our clients, allowing for simple integration into processes for research and development.
Our Goals
At Creative Proteomics, we aspire to be at the forefront of protein analysis and drug characterization. Our mission is to provide industry-leading solutions that empower researchers and facilitate breakthroughs in protein-based therapeutics. Through our comprehensive C-terminal sequencing services, we aim to contribute to advancements in drug discovery, protein engineering, and proteomic research. Please feel free to contact us and we look forward to working with you on attractive projects.
References
- BHAGAVAN, N. V. Protein Isolation and Determination of Amino Acid Sequence. Medical Biochemistry. 2002, 35–50.
- Samyn, B., et al.; A method for C-terminal sequence analysis in the proteomic era (proteins cleaved with cyanogen bromide). Nature Protocols. 2006, 1(1), 318–323.
Related Sections
Services
Applications
For research use only, not intended for any clinical use.
