Creese and Cooper
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The introduction of electron capture dissociation (ECD) [1] in 1998 provided a unique
fragmentation technique for biomolecular analysis. ECD is primarily employed in Fourier
transform ion cyclotron resonance (FT-ICR) mass spectrometers. (It is possible to perform
ECD in an ion trap [2-4] but fragmentation efficiency is not yet comparable to FT-ICR). In
ECD, ions of interest are irradiated with low-energy electrons (≈0.2 eV) producing charge-
reduced radical ions which dissociate via radical driven pathways [5, 6]. ECD differs from
classic tandem mass spectrometry (MS/MS) techniques such as collision induced dissociation
(CID) [7, 8] or infrared multi-photon dissociation (IRMPD) [9, 10]: ECD is believed to be
nonergodic, whereas CID and IRMPD are thermal methods in which the lowest energy bonds
are cleaved first [11]. In ECD, peptide backbone cleavage occurs at N-Cα bonds leading to
c and z∙ (or c∙ and z [12, 13] fragments. In CID and IRMPD, cleavage of N-CO bonds to produce
b and y fragments [14] is observed. There are significant advantages gained by fragmentation
of peptides with ECD: the apparent nonergodic nature of ECD means it is random and relatively
nonselective, the only exception being cleavage N-terminal to proline [15]. The nonselective
nature of ECD means the extent of peptide sequence coverage is greater than for CID [16,
17]. For peptides with labile post-translational modifications (PTMs), loss of the PTM is
usually the dominant fragment observed in CID and IRMPD. In ECD, PTMs are retained on
the peptide backbone [18]. The site of modification can therefore be localized; γ-
carboxyglutamic acid [18], phosphorylation (S, T and Y) [19, 20], N- and O- glycosylation
[21, 22], acylation [23], and sulfation [18] are examples of modifications localized by ECD.
The application of ECD to phosphorylation analysis is of great interest. It is estimated that up
to one-third of all proteins in eukaryotes are phosphorylated [24]. Phosphorylation and
dephosphorylation control complex processes such as signal transduction [25], cell cycle, cell
growth, and metabolism. They also modulate protein activity, stability, interaction, and
localization [26, 27]. The advent of ECD has provided a method for accurately identifying the
sites of modification in phosphopeptides/proteins [19, 20]. CID of phosphopeptides tends to
result in the loss of phosphoric acid H3PO4 (98 Da) from phospho-serine/threonine and (less
so) the phosphate group HPO3 (80 Da) from phosphotyrosine [28] at the expense of sequence
fragments. The loss 98 or 80 Da confirms the presence of phosphorylation; however, in the
absence of sequence fragments, the site can only be located if there is one possible modification
site (S, T, Y) and the sequence of the peptide is known. Because ECD offers greater peptide
coverage and phosphorylation is retained on backbone fragments, the probability of
localization is increased [29]. To date ECD has been applied to the localization of sites of
phosphorylation in the proteins human α-casein [30], cardiac troponin I [31], and Sprouty2
[32], thereby demonstrating its potential for solving “real” biological problems.
Published as: J Am Soc Mass Spectrom. 2008 September ; 19(9): 1263-1274.