Visual proteomics

The ExPASy server (www.expasy.chl is one of the most useful servers, where almost any bioinforma tic tool can be found, together with useful links to other websites such as NCBI or EBI. The several access databases are descriptive, easy to follow, and up to date. Protein data bank searches with SwissProt or Trembl, as well as sequence alignments using either SimAlign (for two sequences) or ClustalW (for more than two protein sequences) can be started from ExPASy, to name just a few of the possibilities available. Access is also given to the Roche Applied Science Biochemical pathways where either keyword searches for particular enzymes or for metabolites can be performed, or entire metabolic pathways or sections thereof can be visualized. Proteomics evaluation is also available on ExPASy, which features free 2D-PAGE software called Melanie. 

Fig. 21.3. Visual proteomics require samples to be prepared by a microfluidic circuit. Cells grown in microreactors are released and sorted to reach a device that lyses them. Various well-known separation circuits are used in series for generating suitable fractions of the lysate. Importantly, a spotting device akin to those used for DNA array production produces 10 pL droplets that are deposited on an EM grid. One cell will provide enough material to cover 1 to a few grids, when one droplet is deposited per grid square...

In addition, initial 3D maps of yet uncharacterized, but reproducibly observed complexes can be obtained from projections of negatively stained samples. Ultimately, the visual proteomics chain will be completed by the inspection of vitrified cell fractions, using cryo-electron microscopy. By sorting out particle projections based on all information established with mass-mapping and 3D reconstruction of negatively stained complexes, high-resolution 3D maps will be obtained. Combined with mass spectrometry data from the respective fractions, these 3D maps will provide a solid foundation for creating atomic scale models of all complexes identified. 

The activity most often associated with proteomics is fractionating and visualizing large numbers of proteins from cells on two-dimensional... 

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

Figure 2.2. Fractionation of protein extracts before 2D gel electrophoresis. Crude lysates can be fractionated by affinity purification or by a number of chromatographic techniques. In addition, organelles or other cellular structures can be purified and lysates from these organelles can be fractionated or separated directly on 2D gels. By repeating this procedure using a number of conditions it may be possible to visualize a large fraction of a cell s proteome.

The dynamic range of protein expression represents a main obstacle since abundant proteins are seldom of interest and others such as transcription factors are only present in a few copies. There is no detector that is able to visualize all proteins at the same time so that prefractionation and the investigation of subproteomes is required. In fact, pre-MS sample preparation techniques exploiting electrophoretic, chromatographic, or chemical properties of the analyte are often the bottleneck of proteomics. 

Precise quantifications are an important quality in molecular biology. There are slight differences in the methods used for global and targeted proteomics. In experiments intended to visualize as many proteins as possible, it is highly desirable to have a parallel quantification method that builds on the display technique. For 2D gel electrophoresis, fluorescent staining methods are under development (Urwin and Jackson, 1993), but they still lack overall sensitivity. Labeling proteins with radioactive isotopes is the most precise method for quantification but is limited to cell cultures, and alternatives are desirable. Recently, a precise method... 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

Staining in proteome analysis is important to visualize proteins in gels. Properties of an ideal 2-DE stain are summarized in Table 5.1. 

Differential 2DE displays of post-gel labelled silver stained and pre-labelled S-35 proteins also represent a sensitive procedure to visualize and identify synthesized elements of proteomes, respectively. 

One of the most useful techniques for visualization of the proteome is two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE). This technique possesses unmatched resolving power for separation of proteins [2-4] and has been used extensively to analyze proteins [5-8], their regulation [9-18], and posttranslational modifications [19-22], Several tech-... 

Functional Profiling and Data Visualization Using Proteomic Data 182... 

Before examining contemporary proteomic workflows, it must be said that the older gel-based technology still remains the largest contributor of proteomic-level information. For many years, the main means to analyze complex protein samples involves separation of proteins on the basis of size and isoelectric point (pi) using two-dimensional polyacrylamide gel electrophoresis (2-DE). The densitometric aspects of gel protein spots provide a direct means to visualize and quantify proteins based on the experimental conditions applied to the... 

The subsequent downstream processing section, which includes visualization of quantified proteins, statistical validation of differences in treatments or samples, and biological interpretation, is much less defined in terms of work-flow regimens and is discussed toward the end of this chapter. In the succeeding text, various relevant aspects of proteomic workflows that impinge on the data obtained from proteomic analysis of prokaryotes assuming that a gel-free approach is used are discussed. 

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