Workflow of C-Terminal Sequencing

C-terminal sequencing, a vital technique in proteomics, identifies the amino acid sequence at the C-terminus of proteins. This method is essential for understanding protein structure and function and has numerous applications in biotechnology and medicine. Below is a comprehensive overview of the workflow involved in C-terminal sequencing.

 

1. Protein Isolation and Purification

The initial step in C-terminal sequencing involves isolating and purifying the target protein. Techniques such as gel electrophoresis, chromatography, and affinity purification are typically employed to achieve a highly pure protein sample, free from contaminants that might interfere with subsequent steps.

 

2. Enzymatic or Chemical Cleavage

Following purification, the protein undergoes enzymatic or chemical cleavage to produce smaller peptides. Enzymes like carboxypeptidases are often used as they sequentially cleave amino acids from the C-terminus. Alternatively, chemical cleavage methods, such as cyanogen bromide (CNBr), target methionine residues to generate peptides with C-terminal fragments.

 

3. Peptide Separation

The resulting peptides are then separated using high-performance liquid chromatography (HPLC) or similar methods. This crucial step isolates the peptides for further analysis, ensuring each peptide is analyzed individually, thus avoiding complications from overlapping sequences.

 

4. Mass Spectrometry Analysis

Mass spectrometry (MS) is integral to C-terminal sequencing. The separated peptides are ionized and analyzed by MS to determine their mass-to-charge ratio. Tandem mass spectrometry (MS/MS) is particularly useful as it provides fragmentation patterns that facilitate amino acid sequence deduction.

 

5. Edman Degradation

In certain cases, Edman degradation is combined with mass spectrometry. This classical method sequentially removes and identifies amino acids from the C-terminus. While more suited for N-terminal sequencing, Edman degradation can offer complementary data for C-terminal analysis when applied judiciously.

 

6. Data Interpretation and Sequence Assembly

Interpreting the mass spectrometry data to assemble the C-terminal sequence is the final step. Software tools and databases assist in matching the mass spectra to known peptide sequences or predicting novel ones. Accurate analysis is critical, especially in differentiating isobaric amino acids (those with identical masses).

 

7. Validation and Verification

To ensure the accuracy of the identified C-terminal sequence, additional validation steps are often required. These might include repeating the sequencing using different cleavage methods or validating the results with synthetic peptides. Consistency across various methods strengthens the reliability of the findings.

 

Applications of C-Terminal Sequencing

C-terminal sequencing finds extensive applications in research and industry. It is crucial for identifying post-translational modifications, elucidating protein degradation pathways, and characterizing recombinant proteins. In pharmaceuticals, it aids in the development of therapeutic proteins and monoclonal antibodies by confirming their correct sequence and structure.

 

By isolating and purifying proteins, generating peptides, and utilizing advanced analytical techniques such as mass spectrometry, scientists can precisely determine the C-terminal sequences of proteins. This information is invaluable for advancing our understanding of protein function and developing new biotechnological applications. MtoZ Biolabs provides integrate C-terminal sequencing service.