De novo protein sequencing is a process by which the amino acid sequence is deduced without prior knowledge of the DNA or protein sequence. This differs from sequence confirmation, where the protein/DNA sequence is already known and the sequence data obtained is used to confirm that it is correct. Therefore, this tool is especially useful to determine the sequence of novel proteins or antibodies, which are not included in the protein database.
De-novo protein sequencing of an intact protein needs careful experimental design, a combination of different analyses and evaluation of data from mass spectrometry, protein chemistry and bioinformatics. First, the matching or similarity between the de novo sequencing peptide and the database search peptide is a good indication that the database search result is correct. Secondly, the de novo sequencing peptides without significant database hits are possibly novel peptides in the sample, and deserve further examination, such as the finding of unexpected PTM or peptide mutations.
The main idea of de novo sequencing is to use the mass difference between two fragment ions to calculate the mass of an amino acid residue on the peptide backbone. Such a process can be continued until all the residues are determined. In a tandem mass spectrometer, the peptide is fragmented along the peptide backbone and the resulting fragment ions are measured to produce the MS/MS spectrum. Depending on the fragmentation methods used, different fragment ion types can be produced. The most widely used fragmentation methods today are Collision-Induced Dissociation (CID) and Electron-Transfer Dissociation (ETD). CID produces mostly b and y-ions; and ETD produces mostly c and z-ions. A good quality spectrum often contains many (but not necessarily all) of the theoretical fragment ions.
In de-novo protein sequence analysis, if one can identify either the y-ion or b-ion series in the spectrum, the peptide sequence can be determined. However, the spectrum obtained from the mass spectrometry instrument does not tell the ion types of the peaks, which require either a human expert or a computer algorithm to figure out during the process of de novo sequencing. During this process, the following factors can cause de novo sequencing to figure out only a partially correct sequence tag from the spectrum: incorrect assignment of y and b ions; some fragment ions are missing; existence of other fragment ion types; existence of noise peaks in the spectrum; the same or similar mass of some residues may cause ambiguity; the PTM (post-translational modifications) on the residues may contribute to the mass ambiguity, as well as complicate the peptide fragmentation pattern.
Advantage of de novo protein sequence analysis service:
Accuracy: We use multiple digestive enzymes to digest the target proteins into overlapping peptides, ensuring all the protein sequences are being covered, as to accurately determine the amino acid sequences.
Comprehensive: Besides protein sequence, we also provide additional analyses on the protein post-translational modifications, S-S bonds, amino acid oxidation and deamination, and so on.
Workflow of our protein sequence analysis service:
1. Digest the protein with multiple enzymes
2. Analyze the digested peptides with LC-MS/MS
3. Computational analyzing protein sequence based on overlapping peptides
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