Even when a database is available, de novo sequencing can contribute to peptide identification in the following ways. This makes de novo sequencing a viable choice for every mass spectrometry analysis in proteomics. However, with recent development in computer algorithms such as PEAKS, speed is no longer an issue. Therefore it has been mostly used when the protein database was unavailable. The following video highlights the de novo sequencing features of PEAKS Studio.ĭe novo sequencing was historically thought to be slow.
#De novo protein sequence analysis manual
Although the basic principle used by computer algorithms is the same as manual de novo sequencing, computer algorithms usually carry out the computation in a very different procedure than manual analysis.įirst released in 2002, PEAKS Studio software has become the industrial standard software for automated de novo sequencing, and is well known for its accuracy, speed, and ease of use. Automated de novo sequencing has been extensively studied in the bioinformatics community and multiple algorithms have been developed. A reliable auto de novo sequencing solution saves precious human time and greatly reduces the labor cost in labs. Manual de novo sequencing requires human experts and is very time consuming. These factors can cause de novo sequencing to figure out only a partially correct sequence tag from the spectrum. The PTM (post-translational modifications) on the residues may contribute to the mass ambiguity, as well as complicate the peptide fragmentation pattern.The same or similar mass of some residues may cause ambiguity (I=L and K=Q).Existence of noise peaks in the spectrum.Existence of other fragment ion types (such as the b 3-NH 3 ion in Figure 1).Some fragment ions are missing (such as b 1 and y 8 in Figure 1).During this process, a few factors can cause difficulties: 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.
#De novo protein sequence analysis series
Thus, if one can identify either the y-ion or b-ion series in the spectrum, the peptide sequence can be determined. A mass table of amino acids is provided for reference. Such a process can be continued until all the residues are determined. Similarly, the next adjacent residue between y 6 and y 5 can be determined as L by the mass difference. For example, the mass difference between the y 7 and y 6 ions in Figure 1 is equal to 129, which is the mass of residue E. The mass can usually uniquely determine the residue. 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. A good quality spectrum often contains many (but not necessarily all) of the theoretical fragment ions. The spectrum consists of peaks at the m/z (mass to charge) values of the corresponding fragment ions. In a CID MS/MS, many copies of the same peptide are fragmented at the peptide backbone to form b and y ions.
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