De Novo Protein Sequencing Procedures and Features

The sequence of a protein molecule is the basis for the function of a protein. Accurate determination of protein sequence plays an important role in the study of protein functional epitopes, detection of commercial monoclonal antibodies, vaccines, and kits. Although there are many studies on protein sequencing, accurate sequencing of proteins is still not as fast and accurate as that of DNA. Protein sequencing is mainly hampered by the purification of protein samples, and the complexity of factors that make up the amino acid composition of proteins.

De novo protein sequencing is also called novel protein sequencing. This technique deduces the amino acid sequence based on the difference in mass between a series of regular fragment ions generated by the peptide collision with an inert gas. We can guess the amino acid sequence and post-translational modifications based on the y ion and b ion at the cleavage of the peptide bond. De novo sequencing has a unique advantage that traditional mass spectrometry sequencing cannot achieve, which is de novo sequencing unknown proteins without relying on any protein database.

In traditional sequencing, it is often the case to detect partial peptide sequences after protein digestion, and then use these peptide sequences to compare with known protein databases. According to certain algorithm scores, proteins and sequences with high probability are calculated.

Limitations of Traditional Sequencing Methods

Mass spectrometry sequencing relies on public databases. If the genome of the species being studied is not fully sequenced or if there is no corresponding sequence in the part, mass spectrometry sequencing will not be able to give a correct identification. The scores and algorithms used by the search engine may also miss the matching of some of the peptides and mass spectra, producing false positive results. If there is a mutation or an unknown modification, the same result cannot be obtained because the search algorithm or parameter settings are not taken into account.

The limitations of these traditional mass spectrometry sequencing may all be overcame in de novo sequencing. Therefore, de novo sequencing can be used to provide accurate assess services that traditional sequencing has not yet been able to offer, including protein mutation analysis, novel protein/peptide de novo sequencing, and mAb sequencing and so on.

Identification Process and Specific Features of De Novo Sequencing