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. This technique is crucial for studying proteins from organisms with unsequenced genomes or proteins with post-translational modifications. Here’s an outline of the experimental principle behind protein de novo sequencing:

Step 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.

Step 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 the re-formation of the disulfide bonds. This step linearizes the protein structure and makes it more amenable to enzymatic or chemical cleavage.

Step 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. This digestion results in a mixture of peptides with known cleavage sites.

Step 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.

Step 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.

Step 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).

Step 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.

Step 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.

Step 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.

Step 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.

De novo sequencing is a complex but powerful technique that enables the exploration and characterization of proteins in the absence of genomic information, thus playing a crucial role in proteomics and related fields of research.

KMD Bioscience has been dedicated to protein expression and sequencing research for over 10 years. Based on high-resolution mass spectrometry, We can provide customers with high-fidelity antibody and protein sequencing services. KMD Bioscience has highly skilled laboratory technicians with extensive experience in protein and antibody sequencing and can provide mass spectrometry-based protein sequencing analysis services. Combined with bioinformatics databases, our sequencing platform can realize rapid and accurate analysis of protein primary structure, and can also provide protein N-terminal sequencing services. For more information, Visit us at to have a detailed understanding.