In order to obtain peptide sequence information by mass spectrometry, fragments of an ion must be produced that reflect structural features of the original compound. Fortunately, most peptides are linear molecules, which allow for relatively straightforward interpretation of the fragmentation data. This is accomplished by colliding the ions with an inert gas. The fragments then monitored via mass analysis.

Tandem mass spectrometry allows for a heterogeneous solution of peptides to be analyzed by filtering the ion of interest into the collision cell, structural information can be derived on each peptide from a complex mixture. The fragment ions produced in this process can be separated into two classes. Once class retains the charge on the N-terminus and fragmentation occurs at a, b, and c. The second class of fragment ions retain the charge on the C-terminus and fragmentation occurs at x, y, and z. Most fragments are obtained from cleavage between a carbonyl and amide bond.

• b ions – charge retained by N-terminus
• y ions – charge retained by C-terminus

In determining the amino acid sequence of a peptide, it is not possible to distinguish between L and I because they have the same mass.

Since a complete ion series (y or b) is not observed, the combination of the two series can provide useful information for protein identification.

Singly charged vs. multiply charged peptides

In multiply-charged peptides, the proton is strongly delocalized. This results in rich fragmentation and good sequence coverage. In singly-charged peptides, the proton is localized on basic residues. This results in poor fragmentation and low sequence coverage.