Instrumentation for LC-MS/MS in Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 56

a single-bond cleavage and so mainly b and y ions are observed
( 33 ) . It also means that if there is a labile bond in the peptide,
for example, the O-phosphate linkage in a phosphopeptide, then
the fragmentation spectrum is dominated by a single fragment ion
( 34 ) , sometimes precluding the ability to identify the peptide.
In quadrupole CID, all ions, precursors and fragments
included, are excited. Hence, products formed by secondary frag-
mentation are also formed. Two backbone cleavages can form
internal ions. In addition, b and y ions can be further fragmented.
The b ion structure is relatively unstable and so readily fragments
further to form smaller b ions ( 35 ) . However, the b2 ion is stable.
Hence, in quadrupole CID spectra, a large percentage of b ions
are fragmented down to a b2 ion giving relatively intense b2 and
a2 ions.
It should also be pointed out that resonant fragmentation
spectra in a quadrupole ion trap lose the low-mass region of the
spectrum, whereas quadrupole CID spectra contain a full mass
range spectrum. This has important implications for certain quan-
titation strategies based on MS/MS peaks, e.g., iTRAQ ( 36 ) , but
this will be discussed in a later chapter.

Electron capture dissociation (ECD) ( 20 ) and electron transfer
dissociation (ETD) ( 14 ) provide orthogonal alternatives to CID
for fragmentation analysis ( 37 ) . Both of these approaches form
unstable radical ions that then fragment at sites that are not the
weakest bonds in the molecular structure. These techniques pro-
duce fragmentation spectra that are less dependent on the peptide
sequence (with the exception of the inability to cleave N-terminal
to proline residues).
ECD involves firing a beam of electrons at the trapped cloud
of sample ions ( 20 ) . Electron capture by the analyte produces a
radical ion, which is unstable and fragments to produce predom-
inantly c and z. ions from peptides. ECD is almost exclusively
performed in FT-ICR instruments.
ETD uses anions, most commonly fluoranthene ions, to
transfer electrons to the analyte, forming radical ions that then
fragment similarly to ECD ( 14 ) . ETD can be performed in
quadrupole ion traps, making the technique much more sensitive
and affordable than ECD in an FT-ICR instrument.
Both of these fragmentation approaches are more effective
on highly charged and charge-dense precursors. As electron cap-
ture or transfer by definition reduces the charge of the precur-
sor, one cannot produce charged fragments from singly charged
components; so ETD/ECD MS/MS analysis of singly charged
species cannot be performed. Also, the efficiency of fragmenta-
tion of doubly charged species is not as high as triply charged or
greater precursors ( 38 ) . Hence, these approaches are not as gener-
ally applicable as CID, but as they are effective on higher charged

3.3.2.ElectronCapture
Dissociation
andElectronTransfer
Dissociation