Current Proteomics - Volume 4, Issue 1, 2007

Almost two decades after the introduction of the electrospray ionization (ESI) and the matrix-assisted laser desorption ionization (MALDI) techniques, mass spectrometry (MS) has become a key technology in the emerging field of proteomics. MS-based procedures combined with various affinity-trapping methods allowed massive identification of constitutive elements in multi-protein complexes from different organisms. Similarly, various MS-based strategies have been developed and applied to low-resolution structural studies on protein and protein complex. In fact, products generated either by limited proteolysis, selective chemical modification or radical probe reactions performed on isolated proteins have been characterized by ESI and MALDI techniques, providing information on the location of residues accessible on molecular surface. Differential experiments performed before and following complex formation allowed the identification of masked regions in each component of the complex after binding, generated as a result of macromolecular interaction. Similarly, MS analysis of protein complex cross-linking products has led to the identification of spatially closed amino acids occurring at molecular interface. Nowadays, these methodologies are used for experimental validation of insilico generated models and verification of interface regions predicted by bioinformatics computations. Accordingly, these approaches present a fundamental resource when severe limitations using high-resolution methods, such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy, which arise from the amount of sample required, difficulties in crystallization or solubilization, may hinder a definitive structural characterization. In this review, the combined use of chemical cross-linking, limited proteolysis, selective chemical modification or radical probe reactions with MS analysis is reviewed and discussed in the point of view of structural biology studies on protein and protein complexes.

Protein-protein interactions play a central role in many cellular processes. Their characterisation is necessary in order to analyse these processes and for the functional identification of unknown proteins. Existing detection methods such as the yeast two-hybrid (Y2H) and tandem affinity purification (TAP) method provide a means to answer rapidly questions regarding protein-protein interactions, but have limitations which restrict their use to certain interaction networks; furthermore they provide little information regarding interaction localisation at the subcellular level. The development of protein-fragment complementation assays (PCA) employing a fluorescent reporter such as a member of the green fluorescent protein (GFP) family has led to a new method of interaction detection termed Bimolecular Fluorescent Complementation (BiFC). These assays have become important tools for understanding protein interactions and the development of whole genome interaction maps. BiFC assays have the advantages of very low background signal coupled with rapid detection of protein-protein interactions in vivo while also providing information regarding interaction compartmentalisation. Modified forms of the assay such as the use of combinations of spectral variants of GFP have allowed simultaneous visualisation of multiple competing interactions in vivo. Advantages and disadvantages of the method are discussed in the context of other fluorescence-based interaction monitoring techniques.

With the completion of genome projects for Arabidopsis thaliana, Oryza sativa and several other plant species, an increasing number of whole genome sequences are now available for plants. In this post-genomic era, a more thorough understanding of gene expression and function can be achieved through the characterization of the products of expression, the proteins, which are essential biological determinants of plant phenotypes. Proteomics offers a continually evolving set of novel techniques to study all facets of protein structure and function. The application of proteomics in plant pathology is becoming more commonplace with techniques such as two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) being used to characterize cellular and extracellular virulence and pathogenicity factors produced by pathogens as well as to identify changes in protein levels in plant hosts upon infection by pathogenic organisms and symbiotic counterparts. This review article summarizes the current status of gel- and non gel-based proteomic techniques and describes the significant discoveries that have resulted from the various proteome-level investigations into phytopathogenic microorganisms and plant host-microbe interactions.

Protein structure and stability rely on the interplay of a large number of weak molecular interactions working in concert to assure a stable and unique native fold. Throughout evolution, different strategies have been devised to modulate protein conformational stability and enhance function and survival of proteins even under adverse conditions. The increasing number of characterized genomes and proteomes, especially those from thermophiles, provides a unique resource to study protein conformations at a wider scale. An integrated proteome-level perspective of protein conformational states in different cellular contexts is likely to contribute to a better understanding of functioning and control of biological systems. This review will address recent proteomic approaches, which allow screening and profiling proteins according to particular conformational features. We will discuss emerging methodologies that allow screening proteomes for unstructured or conformationally altered proteins, and novel approaches that profile and identify proteins within complete proteomes on the basis of their differential resistances to temperature, chemicals, or proteolysis. In particular, the profiling of proteins from thermophiles according to their thermostability will be highlighted as these studies may contribute to elicit general strategies accounting for protein stability and thermostable cellular processes.

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) maps represent an unavoidable tool in many fields connected with proteome research, such as development of new diagnostic assays or new drugs. Unfortunately the information contained in the maps is often so complex that its recognition and extraction usually requires complex statistical treatments. Statistics accompanies many phases of 2D-PAGE maps management - from the spot revelation to maps matching, as well as the extraction and rationalisation of useful information. This review describes and reports the most recent achievements in the field of statistical tools applied to proteome research by two-dimensional gel electrophoresis (2D-GE). The first section is devoted to briefly describe the theoretical aspects of the multivariate methods mostly adopted in this field such as Principal Component Analysis, Cluster Analysis, Classification methods, Artificial Neural Networks. The most recent applications are then described explaining the analysis of spot volume datasets from standard differential analysis as well as the direct analysis of 2D maps images. Applications are also reported about the use of multivariate tools in the analysis of DNA and RNA profiles.