Overview of Quantitative LC-MS Techniques for Proteomics and Activitomics - LC-MS/MS in Proteomics: Methods and Applications - page 34

quadrupole. This analytical strategy can result in very accurate
quantification, especially when using internal standards labelled
with stable isotopes, but even when unlabeled isotopes are not
available and structurally related compounds are used instead,
SRM and MRM techniques are the gold standard for the analysis
of small molecules in analytical chemistry laboratories. The use
of MRM in proteomics is increasingly common ( 161 ) , with some
groups contributing to the systematic identification of optimised
parent to fragment transitions to be used in large-scale quan-
tification of proteomes either experimentally or computationally
( 162 - 166 ) .
Although not canonical, MRM can also be performed in
Q-TOF instruments. As with MRM in triple quadrupoles,
this involves selecting parent ions for fragmentation and then
analysing fragment ions by TOF MS instead of using a second
quadrupole. The disadvantage is that up to 80% of ions are lost
in the orthogonal acceleration region of the Q-TOF configu-
ration, thus resulting in less sensitivity than when using triple
quadrupoles. Also, because longer scan rates are used, Q-TOF
instruments may not be able to monitor as many different ions
simultaneously as triple quadrupoles can. However, MRM in
Q-TOF instruments may also have advantages, including the fact
that since all fragment ions are recorded by TOF, it is possible to
add the signal of several of these fragments in XICs to increase
sensitivity. This may be particularly important in MRM of pep-
tides, where splitting of the signal from the parent ion may trans-
late into a loss in sensitivity. Another advantage is that Q-TOFs
can detect fragment ions with much greater mass accuracy than
triple quadrupoles. This is advantageous and can also result in
enhanced specificity when several isobaric ions co-elute and for
reducing background to enhance signal to noise.
Most modern and competitive proteomics laboratories are
now equipped with triple quadrupoles for MRM quantification
of peptides. This technique is thus being increasingly used for
targeted quantification of several peptides and post-translational
modifications in validation experiments ( 167 ) . However, whether
or not MRM can be used as a general tool for the quantifica-
tion of proteomes in discovery experiments is debatable. This is
because of duty cycle constraints imposed in scanning mass anal-
ysers when switching to the analysis of ions in succession. The
limit of ions that may be quantified in a single experiment by
MRM may be around 500. If internal standards are used and sev-
eral peptides per protein are to be monitored, then this number
may be even lower. Thus at present MRM is restricted to vali-
dation experiments where the aim is to analyse a limited number
of molecules ( 162 - 166 ) . Chapters 9 and 10 provide overviews
of SRM applied to quantitative proteomics and basic design of
MRM experiments, respectively.