De novo peptide sequencing
In mass spectrometry, de novo peptide sequencing is the method in which a peptide amino acid sequence is determined from tandem mass spectrometry.
Knowing the amino acid sequence of peptides from a protein digest is essential to study the biological function of the protein. In the old days, this was accomplished by the Edman degradation procedure. Today, analysis by a tandem mass spectrometer is a more common method to solve the sequencing of peptides. Generally, there are two approaches: database search and de novo sequencing. Database search is a simple version as the mass spectra data of the unknown peptide is submitted and run to find a match with a known peptide sequence, the peptide with the highest matching score will be selected. This approach fails to recognize novel peptides since it can only match to existing sequences in the database. De novo sequencing is an assignment of fragment ions from a mass spectrum. Different algorithms are used for interpretation and most instruments come with de novo sequencing programs.
Peptides are protonated in positive-ion mode. The proton initially locates at the N-terminus or a basic residue side chain, but because of the internal solvation, it can move along the backbone breaking at different sites which result in different fragments. The fragmentation rules are well explained by some publications.
Three different types of backbone bonds can be broken to form peptide fragments: alkyl carbonyl (CHR-CO), peptide amide bond (CO-NH), and amino alkyl bond (NH-CHR).
When the backbone bonds cleave, six different types of sequence ions are formed as shown in Fig. 1. The N-terminal charged fragment ions are classed as a, b or c, while the C-terminal charged ones are classed as x, y or z. The subscript n is the number of amino acid residues. The nomenclature was first proposed by Roepstorff and Fohlman, then Biemann modified it and this became the most widely accepted version.
Among these sequence ions, a, b and y-ions are the most common ion types, especially in the low-energy collision-induced dissociation (CID) mass spectrometers, since the peptide amide bone (CO-NH) is the most vulnerable and the loss of CO from b-ions.
Mass of b-ions = ∑ (residue masses) + 1 (H+)
Mass of y-ions = ∑ (residue masses) + 19 (H2O+H+)
Mass of a-ions = mass of b-ions – 28 (CO)
Double backbone cleavage produces internal ions, acylium-type like H2N-CHR2-CO-NH-CHR3-CO+ or immonium-type like H2N-CHR2-CO-NH+=CHR3. These ions are usually disturbance in the spectra.
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