Electron capture dissociation ECD

In recent years, a novel approach to protein identification emerged, called top-down sequencing. Here the entire nondigested protein is analyzed. Apart from accurate MW measurement, the protein ion is fragmented by the electron capture dissociation (ECD) method (see Chapter 3). This provides in-depth information on the sequence of protein. Such analysis can be performed only with FTICR instruments (see Section 2.2.6) that ensure high resolution and accuracy but, at the same time, they are exceptionally expensive. However, as very large ions are analyzed, even the high accuracy of FTICR is sometimes not sufficient, and it is recommended that such analyses are accompanied by more traditional bottom-up approaches. 

Electron-capture dissociation (ECD) [1] and electron-transfer dissociation (ETD)... 

FT-ICR instruments are also capable of performing MS" experiments. The most popular method of ion activation is sustained off-resonance irradiation (SORI), where ions are excited to a larger cyclotron radius using rf energy, undergo collisions with a neutral gas pulsed into the cell and dissociate. Other methods are available, including infrared multiphoton dissociation (IRMPD)65 and electron capture dissociation (ECD)66 which is of particular value in glyco-peptide analysis (Section VIA). 

Two other ion activation methods were developed to replace the gas molecules as targets by laser beams (photodissociation or infrared multiphoton dissociation IRMPD) or by electron beams (electron capture dissociation ECD). These two methods can be applied to ions that are trapped during their excitations by photons or electrons, respectively. Thus, they are most often used with ion trap or ICR analysers because the residence time and the interaction time are longer. 

Electron capture dissociation (ECD) has recently been developed as an alternative activation method and is now widely used [24,25], The ECD activation method is applied to multiply charged positive ions submitted to a beam of low energy produced by an emitter... 

The fragmentation of peptides can also be obtained by FTICR instruments. Besides the most commonly used activation method, namely CID, the activation can alternatively be performed without gas by infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD). These methods fragment peptide ions in the ICR cell by emitting a laser beam or electron beam, respectively. 

Fragmentation of peptides can also be observed with FTICR instruments. Infrared multiple photon dissociation (IRMPD) and electron capture dissociation (ECD) have been introduced as two alternative dissociation methods to the low-energy CID method. The IRMPD method produces many fragments that make the spectrum very complex and difficult to interpret. Some of the fragment types observed with IRMPD are b and y type ions or these ions that have lost ammonia or water. However, most of them are not these types of fragment ions. 

Electron-capture dissociation (ECD) [89], where the multiple-charge ion captures an electron to produce cationic products. The electron capture results in a radical site in the ion, leading to more and different backbone cleavages in the protein. It has been argued that the fragmentation actually takes place at the electron-capture site, i.e., prior to internal energy redistribution. 

Other fragmentation techniques have been introduced [108]. Some of these, e.g., sustained off-resonance irradiation (SORl) and infrared multiphoton dissociation (IRMPD), provide similar fragmentation as in CID, i.e., preferential backbone cleavages at the peptide amide bond (b- and y-ions). Others like electron-capture dissociation (ECD) [109-110] induce different fragmentation reactions, i.e., the formation of c- and z -ions due to cleavage of N-C bonds. 

To this end, different ion fragmentation tools have been characterized with respect to phosphopeptide fragmentation, e.g., electron-capture dissociation (ECD) [31] and infrared multiphoton dissociation (IRMPD) [32]. An application of ECD in PTM analysis is the top-down protein characterization (Ch. 18.3.5) of carbonic anhydrase [33]. IRMPD is applied in the study on protein kinase C phosphorylation [30]. Both ECD and IRMPD were applied in a subsequent nano-ESI-FT-ICR-MS study on protein kinase A phosphorylation [34]. Combined ECD and IRMPD for multistage MS-MS in FT-ICR-MS was applied for phosphopeptide characterization [35]. ECD provides backbone cleavages (c- and z -ions) without H3PO4 loss, whereas in IRMPD the loss of H3PO4 is prominent and only a few backbone cleavages (b- andy-ions) are observed cf. Ch. 17.6.1). 

The following ion-activation techniques have been used at one time or other to sequence peptides (1) fast atom bombardment (FAB) ionization, (2) CID—tandem MS (MS/MS), (3) ESI in-source CID, (4) MALDI ion-source decay, (5) MALDI postsource decay (PSD), (6) electron-capture dissociation (ECD) and electron-transfer dissociation, and (7) peptide ladder sequencing. Because of the lack of space, only (2) and (4) will be discussed further. 

In all three instrumental configurations, it is possible to induce decomposition of the ion trapped inside the ICR cell not only by collisional experiments, but also by interaction with slow electrons (electron capture dissociation, ECD) or by irradiation with an infrared (IR) laser beam (infrared multi photon dissociations, IRMPD). Experiments of this type (mainly devoted to polypeptide identification) allow to maintain the high-vacuum conditions inside the ICR cell, which are necessary to achieve the high-resolution conditions. 

This novel custom ESI-qQq-FT-ICR instrument allows the performance of several types of MS/MS experiments including Q2 collisionally activated dissociation (Q2 CAD), electron capture dissociation (ECD), and infrared multiphoton dissociation (IRMPD). 

Electron-based dissociation methods (ETD and its forerunner electron capture dissociation, ECD) are highly efficient at generating c- and z -type fragment ions from peptide and whole protein precursor ions via a process that is essentially independent of peptide length, amino acid composition, and post-translation modification (PTM) state [28,45,76-81]. Precursor charge state and m/z-value are important factors, however, that can affect the dissociation efficiency of ETD methods [82-85]. 

Fragmentation of peptide and protein ions in FT-ICR mass spectrometry may be induced by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) [28], infrared multiphoton dissociation (IRMPD) [29,30], blackbody infrared radiative dissociation (BIRD) [31,32], surface-induced dissociation (SID) [33,34], and electron capture dissociation (ECD) [35,36]. These techniques are true MS/MS techniques in which the precursor ion is isolated prior to fragmentation. Additional techniques in which ions are not isolated but fragmented before they... 

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