Peptide Fragmentation

Sequence Ions

The types of fragment ions observed in an MS/MS spectrum depend on many factors including primary sequence, the amount of internal energy, how the energy was introduced, charge state, etc. The accepted nomenclature for fragment ions was first proposed by Roepstorff and Fohlman [Roepstorff, 1984], and subsequently modified by Johnson et. al. [Johnson, 1987].

Fragments will only be detected if they carry at least one charge. If this charge is retained on the N terminal fragment, the ion is classed as either a, b or c. If the charge is retained on the C terminal, the ion type is either x, y or z. A subscript indicates the number of residues in the fragment.

In addition to the proton(s) carrying the charge, c ions and y ions abstract an additional proton from the precursor peptide. Thus, the structures of the six singly charged sequence ion are:

Note that these structures include a single charge carrying proton. In electrospray ionisation, tryptic peptides generally carry two or more charges, so that fragment ions may carry more than one proton.

Internal Cleavage Ions

Double backbone cleavage gives rise to internal fragments. Usually, these are formed by a combination of b type and y type cleavage to produce the illustrated structure, an amino-acylium ion. Sometimes, internal cleavage ions can be formed by a combination of a type and y type cleavage, an amino-immonium ion. Internal fragments are labelled with their 1 letter amino acid code.

Immonium Ions

An internal fragment with just a single side chain formed by a combination of a type and y type cleavage is called an immonium ion. These ions are labelled with the 1 letter code for the corresponding amino acid.

Satellite Ions

Collision induced dissociation of ions at keV energies can generates additional ion types due to side chain cleavages, [Johnson, 1988].

In these structures, R' is the substituent, if any, at the beta carbon following the loss of the gamma carbon by side chain cleavage. Isoleucine and threonine are doubly substituted at the beta carbon, so that side chain loss can give rise to two different ion structures. These pairs are designated d and d' or w and w'.

A useful feature of d and w ions is that they enable the isobaric residues leucine and isoleucine to be differentiated.

Low Energy CID

In low energy CID (i.e. collision induced dissociation in a triple quadrupole or an ion trap) a peptide carrying a positive charge fragments mainly along its backbone, generating predominantly b and y ions. In addition, for fragments containing RKNQ, peaks are seen for ions that have lost ammonia (-17 Da) denoted a*, b* and y*. For fragments containing STED, loss of water (-18 Da) is denoted a°, b° and y°. Satellite ions from side chain cleavage are not observed.

High Energy CID

All of the ion series described above are observed in high energy collision spectra. Relative abundances are composition dependent. Unlike low energy CID, ions do not readily lose ammonia or water.

The characteristic effects of individual amino acid residues on peptide fragmentation behaviour have been reviewed in detail by Papayannopoulos [Papayannopoulos, 1995].

Post Source Decay

The most abundant fragment ion types observed in MALDI-TOF PSD are a, b, and y. If collision gas is used, then the spectra resemble high energy CID. All ion series can be accompanied by composition dependent satellites due to loss of ammonia or water.

When a peptide contains an internal proline, strong ion series due to internal cleavage are observed, extending from the proline in the direction of the C terminus.

ETD and ECD

Electron Transfer Dissociation and Electron Capture Dissociation generate primarily c, y, z+1, and z+2 ions. In some cases, w ions can also be prominent.

Negative Ions

The structures illustrated above are all for ions with a single positive charge. Mascot also includes support for negative ions, and the negative ion types are the same as positive, but with one proton per charge subtracted rather than added. This is based on examples like this. We welcome feedback concerning any additional ion types that should be considered.

Formulae to Calculate Fragment Ion m/z values

[N] is the molecular mass of the neutral N-terminal group, [C] is the molecular mass of the neutral C-terminal group, [M] is molecular mass of the neutral amino acid residues. To obtain m/z values, add or subtract protons as required to obtain the required charge and divide by the number of charges. For example, to get a⁺, add 1 proton to the M_r value for a. To get a^--, subtract 2 protons from the M_r value for a and divide by 2.