Current Proteomics - Volume 1, Issue 3, 2004

The human genome project has opened novel scientific avenues such as structural proteomics. The major challenge in structural proteomics is to predict protein structure-function relationships, including the identification of those proteins whose structures are partially or fully unknown. The use of two-dimensional gel electrophoresis and mass spectrometry methods to identify proteins strongly aids our understanding of biological regulatory networks that govern protein expressions. After identifying proteins, the crucial step is to determine their functions and structures. Recent developments of many high-throughput methodologies and technologies have enabled novel data to be generated with efficiency and speed. Protein structures are typically determined by experimental approaches such as X-ray crystallography or NMR spectroscopy. However, the knowledge of three-dimensional space by these techniques are still limited. Thus, computational methods such as comparative approaches and molecular dynamics simulations are intensively used as alternative tools to predict the three-dimensional structures and dynamic behaviors of proteins. This review summarizes recent advances in high-throughput structural proteomics that involve instrumentation methods such as two-dimensional gel electrophoresis, mass spectrometry, X-ray crystallography, and NMR spectroscopy, and computational methods such as comparative approaches and molecular dynamics simulations. New insights into proteinprotein interactions and relationships between structure and protein-protein interactions will also be presented.

Protein arrays with ability to simultaneously detect multiple protein expression levels, protein-protein interactions, protein-DNA or RNA interactions, protein-small molecule interactions and protein modifications will be an excellent tool in biomedical discovery. A variety of technologies have been developed to meet this need. In general, these assay systems can be divided into two major categories: label-based approaches, and sandwich or enzyme linked immunosorbent assay (ELISA)-based approaches. The purpose of this review is to provide an overview of the principles and techniques of ELISA-based protein arrays, with particular emphasis on the use of ELISA-based protein arrays for detection of multiple protein expression levels.

The formation of biofilms is a universal bacterial survival strategy. Biofilms occur on inert and living supports in natural environments and in industrial installations. Given their importance in various industrially relevant areas and in human health, numerous investigations have focussed on the particular physiology of these fixed microorganisms. It is now well recognised that bacteria present in biofilms behave quite differently from their planktonic counterparts. In particular, biofilm organisms are far more resistant to antimicrobial agents than are planktonic organisms. The mechanisms involved in the resistance of biofilm bacteria to antimicrobials are complex and still not fully understood. One of the hypotheses that suggested to explain the increased resistance of biofilms to antimicrobial agents assumes the existence of significant differences in gene expression. Although the expression of a limited number of genes appears to be altered during biofilm growth, a number of proteomics studies have revealed large physiological differences between free-living and biofilm bacteria. Moreover, multiple phenotypes were identified during the different stages of biofilm development. This review presents recent data on protein expression in sessile microorganisms that support the existence of a specific metabolic behaviour of biofilm bacteria.

With the advent of proteomic methods, a sudden switch from genomics to proteomics has been observed and indeed, in many cases no definite conclusions can be drawn from the nucleic acid data. Moreover, there is a long and unpredictable way from RNA to protein and differences between neurodegenerative disorders at the mRNA level could not be verified at the protein level. However, proteins carry out the ultimate function and thus the role of proteins is pivotal. Two-dimensional gel electrophoresis with in-gel digestion followed by mass spectrometric methods and subsequent quantification of proteins with specific software allow study of protein expression in a high-throughput way, identifying large series of proteins concomitantly. Furthermore, evaluation of protein expression by proteomic technologies does no longer rely upon the availability and specificity of antibodies. Significant proteomic approaches have been used to study degenerative diseases, including Alzheimer's disease, and many protein classes from signalling, metabolic, cytoskeleton, chaperone, antioxidant, and proteasome have been shown to be tentatively involved in pathological mechanisms. The aim of this review is to show what has been studied so far in the area using proteomic approaches, describing methodologies used including their potentials and limitations, and finally what still remains to be done. We are addressing only those methods and results necessary for discussing proteomic observations. The message of this review is that proteomics carries the potential to find important clues for pathogenesis of neurodegenerative diseases and to identify pharmacological targets, once the crucial problems such as analysis of hydrophobic and membrane proteins are solved.

Fertilization is the biological bridge between gametogenesis and embryogenesis, by which sperm and eggs grow and mature, mate, and fuse with each other to give rise to the birth of a new individual(s). In this review, we address the current status of the knowledge in the field of fertilization, with a special focus on the role of maternal and paternal protein molecules such as tyrosine kinases, phospholipase C, and inositol trisphosphate receptor / Ca2+ channels in the establishment of intracellular Ca 2+ release, block to polyspermy, transition from meiotic to mitotic cell cycle, and nuclear fusion that are collectively called “sperm-induced egg activation”. We then discuss some proteomics approaches to analyze signaling events associated with sperm-induced egg activation. Protein profiling of subcellular microdomains and its differential display before and after fertilization, screening of fertilization-specific antigens by using a subtractive immunization scheme, and in vitro reconstitution of egg activation events using cell-free systems are now under investigation in our research team. These new experimental approaches, collectively termed “fertilizome project”, will be useful to understand the complexity of fertilization signaling, and will also be necessary for understanding and improving assisted reproduction and reproductive cloning techniques.

This review covers the vast array of methods that appeared in the last few years for separation and identification of thylakoid membrane proteins present in chloroplast, a good model for setting up analytical methods suitable for membrane proteins. Although the major method for protein separation is gel electrophoresis (GE), we will summarise in this review the results of several studies performed to develop a rapid and straightforward method based on high-performance liquid chromatography (HPLC) to resolve most of the antenna proteins of the two photosystems, photosystem I (PSI) and II (PSII) and identify them by intact molecular mass determination using electrospray ionisation mass spectrometry (ESI-MS). The complex mixtures of these strongly hydrophobic membrane proteins are prefractionated by sucrose gradient ultracentrifugation in the first dimension followed by reversed-phase HPLC in the second dimension. The separation achieved is by far superior to that possible with GE. It is based on small differences in hydrophobicity of these very similar proteins. The correspondence between the intact molecular masses measured with those deduced from the DNA sequence enabled the identification of the different protein components of antenna complex. Isomeric forms of the proteins were readily revealed. On the contrary, peptide mass fingerprinting, commonly used for soluble proteins, does not seem to be very helpful for identification of these highly hydrophobic membrane proteins due to the limited number of peptides which can be obtained by proteolysis. Thus, separations and identifications of antenna proteins presented in this study may serve as reference for confident identification in future studies dealing with any membrane proteins.