Biology Reference
In-Depth Information
with fragments still analyzed in the mass analyzer, e.g., Orbitrap or
Time of Flight. This type of fragmentation is also referred to as
higher
energy collision dissociation
(HCD) fragmentation and more closely
resembles the fragmentation pattern observed in a triple quadrupole
instrument and is therefore the method of choice to obtain SRM
assays directly from shotgun experiments [
12
]. Typically, multiple
transitions of a HCD spectrum are selected and the same sample ana-
lyzed on a triple quadrupole instrument. The fi ve most intense transi-
tions are then used for a high confi dence SRM assay.
Obtaining SRM assays from HCD shotgun experiments is very
cost effi cient. The drawback of this method is that SRM assays are
obtained only for peptides that are detectable by shotgun mass spec-
trometry. If the protein(s) of interest are not in the list of identifi ed
peptides, fractionation is necessary. This fractionation can be in form
of biological fragmentation of cellular subcomponents, or enrichment
of proteins and/or protein complexes using affi nity purifi cation mass
spectrometry [
13
]. Another way of fractionation is to separate pro-
teins or peptides on the basis of their physical properties, e.g., fi rst
separate peptides by off-gel isoelectric focusing (pI) [
14
] and then
analyze each fraction using shotgun reverse phase LC-MS/MS run.
As additional fractionation steps are labor intense and require sev-
eral LC-MS/MS runs, another avenue are chemically synthesized
peptides. Very small peptide amounts are already suffi cient to gen-
erate SRM assays either in HCD shotgun mode or using mass
spectrometers with a trapping device in MS1 triggered MS2 mode.
The initial peptide spectra are then confi rmed in SRM mode in the
triple quadrupole instrument.
There are several advantages to this method: chemical peptide
synthesis up to 25 amino acids is very cost effi cient and many pep-
tides can be analyzed simultaneously. As all chemically synthesized
peptides are targeted peptides, resources are used effi ciently, com-
pared to off-gel pre-fractionation discussed above. Another advan-
tage: peptide sequences can be synthesized and SRM assays obtained
for peptides not detected by shotgun mass spectrometry before
[
15
]. The disadvantage of the method lies in the prediction of pep-
tides to be synthesized. There are many selection criteria: peptides
already detected in other shotgun experiments; proteotypic pep-
tides; predicted favorable electrospray mass spectrometry proper-
ties; no or little theoretical shared transitions; favorable hydrophobic/
hydrophilic properties; fully tryptic peptide [
16
]. The latter crite-
rion dictates that only a Lys or Arg residue resides at the C-terminus
of a peptide. Due to diffi culties predicting precise protease cleavage
activity, semi-tryptic peptides are typically not considered. Further,
once a choice is made to use a certain protease, e.g., trypsin cleaving
C-terminal to Lys or Arg, in the future new peptides have to be
synthesized if another protease is used, e.g., Asp-N cleaving
N-terminal to Asp or Glu. An additional protease digest might be
2.2.3
Synthetic Peptides
