Peptide de novo sequencing is the analytical process that derives a peptide’s amino acid sequence from its tandem mass spectrum (MS/MS) without the assistance of a sequence database. It is in contrast to another popular peptide identification approach – “database search”, which searches in a given database to find the target peptide. A clear advantage of de novo sequencing is that it works for both database and novel peptides.

Basic Principle

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 (Figure 1). 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.

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. The mass can usually uniquely determine the residue. For example, the mass difference between the y₇ and y₆ ions in Figure 1 is equal to 129, which is the mass of residue E. Similarly, the next adjacent residue between y₆ and y₅ can be determined as L by the mass difference. Such a process can be continued until all the residues are determined. A mass table of amino acids is provided for reference.

Thus, 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, a few factors can cause difficulties:

• Incorrect assignment of y and b ions.
• Some fragment ions are missing (such as b₁ and y₈ in Figure 1).
• Existence of other fragment ion types (such as the b₃-NH₃ ion in Figure 1).
• Existence of noise peaks in the spectrum.
• The same or similar mass of some residues may cause ambiguity (I=L and K=Q).
• The PTM (post-translational modifications) on the residues may contribute to the mass ambiguity, as well as complicate the peptide fragmentation pattern.

These factors can cause de novo sequencing to figure out only a partially correct sequence tag from the spectrum.

With a Database

De novo sequencing was historically thought to be slow. Therefore it has been mostly used when the protein database was unavailable. However, with recent development in computer algorithms such as PEAKS, speed is no longer an issue. This makes de novo sequencing a viable choice for every mass spectrometry analysis in proteomics. Even when a database is available, de novo sequencing can contribute to peptide identification in the following ways.

1. 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. Therefore, de novo sequencing can be used to improve database search performance.
2. 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.

These considerations have been employed in the standard workflow of PEAKS Studio as illustrated in Figure 2.