Biology Reference
In-Depth Information
sophisticated software suites are used to extract 'real' protein signals and to discrim-
inate them from artificial signals such as image noise, fluorescent speckles and other
disrupting features. Following the detection of genuine signals, the images are pro-
cessed to generate consensus spot patterns of all the images within the experiment
( Luhn et al. , 2003 ). For this purpose, the signal intensities of all gel images are com-
bined to generate an artificial proteome map from which the spot boundaries are
determined. Matrix assisted laser desorption/ionisation based mass spectrometry
is then used to identify the proteins in as many of the detected spots of the proteome
map as possible. With these spot identifications at hand, complemented by the estab-
lished spot boundaries of the proteome map and a series of positionally corrected 2D
gel images, it is possible to extract comprehensive, relative expression profiles.
6 LARGE-SCALE ABSOLUTE QUANTITATIVE PROTEOMICS
WITH SRM-CALIBRATED 2D PAGE
As introduced above, several approaches have been developed for large-scale abso-
lute quantification of proteomic data. All of these approaches are suitable for the
quantification of the soluble proteins mostly found in the cytosolic cell extracts,
effectively guaranteeing a reproducible proteome analysis for this cellular sub-
fraction of proteins. Although in part these methods generate their data in markedly
differing ways, theoretically, the results should be comparable. Some of the methods
rely heavily on high-end mass spectrometric equipment, whilst others use relatively
basic and widely available equipment. Consequently, there is a trade-off between
methods that provide very precise quantitative data, but at a high cost, and those
in which the same level of precision cannot be achieved, but involve considerably
less effort and resources. Here, we consider a combination of the classical proteomics
approach to 2D differential image analysis as a well-established and relatively
cost-effective method for analysing complex proteome samples, and a targeted
proteomics approach based on SRM and relatively basic mass spectrometric equip-
ment. Having already introduced the methodologies for SRM analyses based on
AQUA and QconCAT, as well as differential 2D gel image analysis, we now focus
on the steps necessary to integrate these methods to quantify soluble protein separated
by 2DE.
In a study relying on SRM-calibrated 2DE gels, all quantitative aspects of sample
preparation have to be tracked, including cell count, cell size/volume, efficiency of
cell disruption, determination of protein content and compensation for protein loss at
different preparation steps by using spiked-in protein standards from the beginning.
All these steps are summarized in Figure 3.1A . With this extensively characterized
protein sample at hand, two branches of the proteome workflow have to be followed,
as shown in Figure 3.1B .
In the first place, a 2DE analysis is carried out for all proteome samples under
investigation. As a result, relative protein expression profiles are generated that
can be used subsequently for calibration with the targeted proteomics part of the
Search WWH ::




Custom Search