Biology Reference
In-Depth Information
as SSR-Calc [
27
]. However, there are discrepancies between in
silico predicted retention times of peptides and experimentally
observed retention times [
29
]. Further, the retention time of a
peptide was already empirically determined during the initial
screening phase (
see
Subheadings 2.2.2/2.2.3/2.2.4), which is a
more accurate measure than in silico prediction. In order to uni-
versally apply the measured retention times of targeted peptides
to various LC-gradients and MS setups, reference peptides are
also measured during the same run and relative retention times
determined [
29
].
In detail, the reference retention time peptides span the
whole range of the LC-gradient and a linear regression is cal-
culated onto which the targeted peptides are projected. Once
these relative values have been established, the same relative
retention time peptides can be measured on different LC-MS
systems allowing for a robust detection of targeted analytes
[
29
]. Principally there are two ways of establishing relative
retention times: using an external standard (multiple vendors
sell retention time peptides) or internal standards (using pep-
tides of “housekeeping” proteins). Latter approach has the
advantage of not adding any additional peptides to a precious
sample, but “housekeeping” proteins might differ depending
on tissue type and/or species analyzed.
One such implementation of relative retention times is iRT
[
29
]. There, eleven reference peptides were synthesized which
do not share any peptide sequence with currently known pro-
teins and elute off a C18 column during the entire gradient
due to their diverse hydrophobic properties. Even measuring
only seven reference peptides will decrease retention time pre-
cision by only 10 %. These retention time peptides are mea-
sured with three transitions each and their retention time is
monitored (Table
2
).
4. Sample Preparation
The advantage of SRM-MS lies in the fact that targeted pro-
teomics allows for consistent monitoring of analytes from
unfractionated lysate. Hence, special care should be taken to
obtain peptides well suited for nano-LC systems allowing for
multiple subsequent injections of peptide samples. Depending
on the species under investigation, e.g., angiosperms or gym-
nosperms [
30
], different lysis protocols will be employed [
31
,
32
]. As traditional detergents used for cell lysis are not com-
patible with LC-MS in general, specialized protocols were
established [
33
,
34
] or specialized LC-MS compatible deter-
gents were developed. One such specialized detergent is
Sodium3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-
1-propanes-ulfonate (World patent number 2005116607),
also known under the trade mark RapiGest™ SF (Waters
