Analysis of Post-translational Modifications by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 102

production of an acetylated lysine immonium ion at m/z 143.1
and the related ion (demonstrating loss of NH 3 )at m/z 126.1
( 56 , 57 ) .

A neutral loss scan determines the m / z of parent ions which frag-
ment to produce a product ion demonstrating loss of a specified
(neutral) mass. The resultant product ion generated following this
neutral loss can then be selected for further fragmentation in an
MS/MS/MS experiment. Neutral loss experiments of this sort
can be achieved only on those instruments capable of multiple
stages of tandem MS, namely ion-trapping instruments. Modi-
fied peptides which demonstrate such a PTM-specific loss during
CID can easily be characterized using this technique. However,
as is the case with phosphorylated peptides, direct mapping of the
site of modification may be difficult. In addition to performing
an MS/MS/MS CID experiment, the observation of a neutral
loss during CID can also be used to trigger ECD or ETD of
the precursor of interest; this is particularly beneficial for those
labile precursors for which sequence-determining fragment ions
are difficult to generate ( 58 ) . Combining ETD data generated
in this manner with CID information could prove particularly
useful for improving confidence in the identification of sites of
phosphorylation, glycosylation, and methylated arginine amongst
others ( 59 ) .

4.2.NeutralLoss
Scanning

4.3.Selected
Reaction
Monitoring/Multiple
ReactionMonitoring

Selected reaction monitoring (SRM) and multiple reaction moni-
toring (MRM) rely on the fragmentation of a specific peptide ion
to yield one or more defined product ions which are diagnostic
for a specific series of amino acids. In SRM, a product ion of spec-
ified m / z derived from a particular precursor ion m /z window is
used to confirm the presence of a specific analyte. This is extended
with MRM, where a number of different fragment ion m/z val-
ues are used to confirm the presence of peptide ions of inter-
est, providing greater confidence in the assignment. Due to the
increase in sensitivity of identification and quantification achieved,
SRM and MRM can be used in the identification of modified
peptides derived from a known post-translationally modified pro-
tein. This technique, adapted under the acronym “multiple reac-
tion monitoring-initiated detection and sequencing” (MIDAS),
has been used successfully for the identification of sites of phos-
phorylation and ubiquitination from purified proteins containing
known modifications ( 60 , 61 ) .

5. Quantification
of PTMs

Elucidation of the stoichiometry of a given modification is impor-
tant for correlation with the proportion of a protein exhibiting
altered PTM-regulated functionality, be that an enzyme activity