Biology Reference
In-Depth Information
treated with care with respect to their low sample volumes, the need for fast cooling
and the rapid removal of residual buffers/media originating from the growth
medium. Furthermore, the determination of low MW intracellular and extracellular
compounds, as analyzed in metabolome studies, requires the rapid stabilization of
their physiological status because metabolite pools tend to be highly dynamic. Addi-
tionally, compounds often found in defined growth media, which are known to cause
quenching effects, can affect metabolome studies. Consequently, the experimental
design starts as early as the choice for media for cultivation and becomes even more
important at the sampling and subsequent processing stages.
3.1.2
Cultivation
Systems biology approaches depend on robust protocols for bacterial cell growth.
To achieve this, appropriate cell growth media and cultivation conditions have to
be tested for robustness and reproducibility. Key cornerstones are the standardized
preparation of stock cultures and the use of standard operation procedures for the
pre- and main cultures, ensuring little variation in the growth rate as a measure of
reproducibility.
3.1.3
Determination of cell counts
Absolute proteomic studies require the number of cells in a sample to be accurately
determined at each sampling time. Various methods are available including deter-
mining the number of colony-forming units and measuring optical density using a
spectrophotometer. In our hands, the determination of cell numbers in a counting
chamber has proved to be the gold standard. Unlike methods that rely solely on the
density of a suspension, visual counting allows live and dead cells to be distinguished,
as well as the detection of unusual cell morphologies. Photographing a series of
counting chamber grids helps to shorten the time required for the analysis and helps
keeping the cells in the physiological state of interest.
3.1.4
Determination of cell dimensions
The estimation of cell dimensions (e.g. average cell length, cell diameter and cell
wall/membrane thickness) is essential for calculations based on the cell volume.
These values are determinants of cell morphology and are dependent on the
FIGURE 3.1
(A) Preparation of cell extracts for absolute quantification strategies in proteomics. The key
features/steps of the preparation of cell extracts for absolute quantitative proteomics for use in
systems biology approaches. (B) Large-scale absolute quantitative proteomics with SRM-
calibrated 2D gels. Outline of the complete workflow of SRM-calibrated 2DE. The left-hand
column shows the workflow for 2DE part of the analysis, leading to the determination of
anchor proteins. The workflow leading to the large-scale absolute quantification of the anchor
proteins by targeted analysis based on AQUA (middle column) or on QconCAT (right-hand
column).
