Biology Reference
In-Depth Information
workflow. Working under the assumption that all proteins behave similarly with
respect to their entry into the gel and their binding of fluorescent dye, the use of cal-
ibration proteins which are spiked into the samples before 2D separation facilitate the
quantitative detection of all of the resolved proteins. Normally, the spot volumes of
such calibration proteins would be calculated and expressed as a proportion of total
protein extract, as determined from the sum of the individually resolved protein
spots. This relative quantitative estimation can then be used to calculate protein
amounts independently of the fractional losses of protein extracts during the 2DE
process.
In the SRM-calibrated 2D gels, another path is followed: a subset of proteins is
chosen from the differential 2D gel image to serve as 'anchor proteins'. These anchor
proteins serve as the internal standards in the complete analysis: instead of spiking-in
known amounts into the sample as indicated above, the absolute concentration in the
sample is determined by targeted SRM analyses. To be a candidate anchor protein,
it has to be present on all the 2D gels examined. This is especially important in
proteome studies that examine the influence of certain triggers, such as antibiotic
stresses, that lead to marked rearrangements of the cell's protein inventory. In this
case, a characteristic of a proteome study, namely, the relative stability of the
proteomic fraction compared to the transcriptome, represents an advantage. Whilst
a number of proteins undergo rapid degradation by a combination of proteases and
the cell's quality control system, even under starvation conditions, a significant
proportion of proteins remain relatively stable in a time frame that can be measured
in hours (
Becher
et al.
, 2009; Otto
et al.
, 2010; Michalik
et al.
, 2012
). Consequently,
proteins that fulfil this criterion will be available in most microbial proteomic
studies.
Putative anchor proteins have to form good quality spots that are well separated
from other spots on the gels. Sufficient resolution will lead to unambiguous and reli-
able detection by commercial software packages, complemented either by direct
mass spectrometric identification or by spot matching with a pre-assembled master
gel/proteome map. To ensure bias-free calibration, the selected anchor proteins
should cover the entire range of molecular masses and isoelectric points to be exam-
ined on the 2D gels. These criteria are possible in most cases. Proteins that reside in
multiple spots or that show a high degree of variability are difficult to quantify reli-
ably and should not be selected as anchor proteins. Such proteins are likely to have
undergone PTM or to be targets of protease activity.
After relative quantification of all proteins covered by the 2DE analysis, and, in
particular, the anchor proteins, the absolute protein concentrations of the anchor pro-
teins are determined by SRM as previously described (
Figure 3.1B
). Combining the
absolute data from the SRM analysis with the relative data from the differential 2D
gel image analysis is possible using two approaches. In the first approach, each pro-
teome sample, and therefore each single gel, is calibrated by itself. This is referred to
as intragel calibration. In the second approach, the whole experimental set-up allows
for the calibration of only a part of the proteome samples (e.g. a single time point
within a complete time series or a control sample in a dose-dependent study) referred
