Biology Reference
In-Depth Information
2.3
Large-scale absolute quantification in proteomics
Isotope dilution-based absolute quantification strategies are perfectly suited for the
determination of small (PSAQ, AQUA) to medium (QconCAT) scale protein
quantities, for example, of enzymes of specific biochemical pathways. Although
Brownridge
et al.
(2011) have applied the QconCAT method on a large scale in
yeast, scaling up of these methods is, in most cases, restricted by the costs of the pep-
tides required if hundreds or even thousands of proteins are targeted for analysis or
simply by the time that has to be spent on QconCAT design, the optimization needed
for each specific peptide assay and the time needed for data acquisition. Therefore,
a number of studies have strived to combine large-scale proteomics data with abso-
lute protein quantification with the aim of yielding absolute proteome data on a large
scale (
Malmstr¨m
et al.
, 2009; Maass
et al.
, 2011; Maier
et al.
, 2011; Schmidt
et al.
,
2011
). To achieve this, large-scale proteome datasets are first acquired either by
mass spectrometry-based or by 2D gel-based proteomics. Subsequently, relative
quantification is carried out for all samples in the study. This could be based on stain-
ing intensities in 2D gel-based approaches or on integrated peak signals in mass
spectrometry-based approaches. For calibration of a large-scale dataset, absolute
amounts of a small subset of proteins, called anchor proteins, are determined by tar-
geted proteome analysis using either spiked-in heavy labelled peptides (AQUA) or
peptides generated by QconCAT. The absolute quantification data from the anchor
proteins are then propagated to all of the other measured proteins.
The label-free methods relying on the 'best flyer' concept are,
per se
, putative
large-scale methods. Here, the proteome coverage is restricted by the capacity of
the LC-MS systemused for the analysis. In viewof the relatively easy implementation
and the achievable high-throughput, label-free methods appear to be more attractive
for large-scale studies. However, it should be noted that these methods are still less
precise than methods based on stable isotope dilution (
Bantscheff
et al.
, 2012
).
3
THE PROTEOMICS WORKFLOW
3.1
Sample preparation for absolute quantification strategies
in proteomics
3.1.1
General considerations
Despite major methodological differences in absolute quantification strategies, the
methods for preparing cell extracts are comparable, with only minor changes applied
for specific workflows (
Figure 3.1A
). In general, to fulfil the requirements of differ-
ent 'omics' techniques, the complete design in the workflow, including, for example,
sample collection, storage and processing, must be coordinated throughout the whole
experiment. Technical issues may be encountered when combining transcriptome,
proteome and metabolome sampling in parallel. Whilst sampling for transcriptome
studies requires care to avoid RNA degradation (e.g. inclusion of inhibitors and rapid
cooling/killing of cells by harvesting on ice-cold buffer), proteome samples are
