When the precursor cations (proteins or peptides) and radical anions are combined in the ion trap an electron is transferred to the mulitply-charged cation. This forms an unstable positive radical cation with one less positive charge and an odd electron. Fragmentation takes place along the peptide backbone at a N− Cα bond, resulting in c- and z-type fragment ions.<ref name=":1" />
When the precursor cations (proteins or peptides) and radical anions are combined in the ion trap an electron is transferred to the mulitply-charged cation. This forms an unstable positive radical cation with one less positive charge and an odd electron. Fragmentation takes place along the peptide backbone at a N− Cα bond, resulting in c- and z-type fragment ions.<ref name=":1" />
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Electron-capture dissociation (ECD) was developed in 1998 to fragment large proteins for mass spectrometric analysis.[6] Because ECD requires a large amount of near-thermal electrons (<0.2eV), originally it was used exclusively with Fourier transform ion cyclotron resonance mass spectrometry (FTICR), the most expensive form of MS instrumentation.[7] Less costly options such as quadrupole time-of-flight (Q-TOF), quadrupole ion trap (QIT) and linear quadrupole ion trap (QLT) instruments used the more energy-intensive collision-induced dissociation method (CID), resulting in random fragmentation of peptides and proteins.[8] In 2004 Syka et al announced the creation of ETD, a dissociation method similar to ECD, but using a low-cost, widely available commercial spectrometer. The first ETD experiments were run on a QLT mass spectrometer with an electrospray ionization (ESI) source. [9]
Principle of operation
Several steps are involved in electron transfer dissociation. Usually a protein mixture is first separated using high performance liquid chromatography (HPLC). Next multiply-protonated precursor molecules are generated by electrospray ionization and injected into the mass spectrometer. (Only molecules with a charge of 2+ or greater can be used in ETD.) In order for an electron to be transferred to the positive precursor molecules radical anions are generated and put into the ion trap with them. During the ion/ion reaction an electron is transferred to the positively-charged protein or peptide, causing fragmentation along the peptide backbone. Finally the resultant fragments are mass analyzed.[10]
Radical anion preparation
In the original ETD experiments anthracene (C14H10) was was used to generate reactive radical anions through negative chemical ionization.[9] Several polycyclic aromatic hydrocarbon molecules have been used in subsequent experiments, with fluoranthene currently the preferred reagent.[11] Fluoranthene has only about 40% efficiency in electron transfer, however, so other molecules with low electron affinity are being sought. [10]
Injection and fragmentation
When the precursor cations (proteins or peptides) and radical anions are combined in the ion trap an electron is transferred to the mulitply-charged cation. This forms an unstable positive radical cation with one less positive charge and an odd electron. Fragmentation takes place along the peptide backbone at a N− Cα bond, resulting in c- and z-type fragment ions.[3]
Mass analysis
Instrumentation
Electron transfer dissociation takes place in an ion trap mass spectrometer with an electrospray ionization source.
Proteomics
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Changes to the method
newer applications including characterization of PTMs, non-tryptic peptides and intact proteins. (Kim Review)
^US patent 7534622, Donald F. Hunt, Joshua J. Coon, John E.P. Syka, Jarrod A. Marto, "Electron transfer dissociation for biopolymer sequence mass spectrometric analysis", issued 2009-05-19