Principle of protein de novo sequencing

TIANJIN, China - Dec. 5, 2023 - PRLog -- De novo sequencing refers to the process of determining the amino acid sequence of a protein from scratch, without any prior knowledge of its DNA sequence. Here's an outline of the experimental principle behind protein de novo sequencing:

1: Protein Isolation and Purification:
The first step involves isolating the target protein from a complex mixture and purifying it to a sufficient degree to allow for accurate sequencing.

2: Protein Reduction and Alkylation (optional):
If the protein contains disulfide bonds, they may be reduced to free thiol groups and then alkylated to prevent re-formation of the disulfide bonds.

3: Protein Digestion:
The purified protein is enzymatically or chemically digested into smaller peptide fragments. Common proteolytic enzymes used for digestion include trypsin and chymotrypsin, which cleave proteins at specific amino acid residues.

4: Peptide Separation:
The resulting peptide mixture is often separated using chromatographic techniques such as liquid chromatography (LC) to reduce complexity and enable more accurate sequencing.

5: Mass Spectrometry Analysis:
The separated peptides are analyzed by mass spectrometry (MS). The mass spectrometer measures the mass-to-charge ratio of the peptides and their fragments, generating a spectrum that reflects the peptide's composition and sequence.

6: Peptide Fragmentation:
Within the mass spectrometer, peptides are fragmented along the backbone, generating a series of ionized fragments. This process is often achieved through techniques like Collision-Induced Dissociation (CID) or Electron Transfer Dissociation (ETD).

7: Spectrum Analysis:
The generated spectra are analyzed to determine the sequence of amino acids in each peptide. The analysis involves matching the observed fragment ion masses to theoretical fragment ion masses derived from all possible amino acid sequences.

8: Sequence Assembly:
The amino acid sequences of individual peptides are assembled to deduce the complete sequence of the original protein. Overlapping regions between peptides help in accurately assembling the full sequence.

9: Database Searching and Sequence Alignment (if applicable):
While de novo sequencing is intended for proteins with unknown sequences, any obtained sequence information can be compared with known sequences in databases to find homologies or to confirm the de novo sequencing results.

10: Validation:
Additional experiments or analyses may be conducted to validate the obtained protein sequence, such as synthesis and characterization of the protein based on the deduced sequence or comparison with similar known proteins.

KMD Bioscience has highly skilled laboratory technicians with extensive experience in protein and antibody sequencing, and is able to provide mass spectrometry-based protein sequencing analysis services. For more information, Visit us at https://www.kmdbioscience.com to have a detailed understanding.