Current Proteomics - Volume 4, Issue 2, 2007

Proteins are the major players in most processes of living cells and study of the proteome has great relevance to investigations on cells and organisms at the molecular level. 2-DE is a core and powerful proteomic technique to study protein expression and function in living organisms and it allows a fast overview of changes that occurred in cells at the proteome level. Although data on DNA sequences from large scale genomic sequencing projects has greatly facilitated the identification and characterization of proteins, genomic sequences of many organisms, including that of many microalgae are still unknown. Thus, de novo protein/peptide sequencing techniques are important for acquiring amino acid sequences of proteins from organisms with incomplete genome data. Take dinoflagellates as an example of micro-algae for discussion purposes, they are the major causative agents of harmful algal blooms (HABs). Lack of well described genome information of dinoflagellates has limited progress of proteomic studies on these organisms. Nevertheless, 2-DE combined with de novo protein/peptide sequencing could provide an alternative route for identification and annotating proteins of interest in these organisms. However, preparation of high-quality samples of dinoflagellate cell extracts for 2-DE analysis is difficult due to the presence of high endogenous levels of nucleic acids, polysaccharides, salts, pigments, and other interfering compounds. Thus, an optimized protocol of sample preparation is a pre-requisite for obtaining satisfactory and reproducible 2-DE profiles. In light of these perceived problems, we had reviewed current methods available for preparing dinoflagellate samples for 2-DE analysis with some working alternatives from the authors' laboratory. Moreover, de-novo protein/peptide sequencing methods by MALDI-TOF MS assisted by effective derivatization protocols were also described.

Phytopathogenic fungi are organisms responsible for several plant diseases in different crops around the world, causing very important economic losses to the farmers. Fungi have a complicated life cycle, normally with asexual and sexual reproduction that involves the formation of different reproductive structures. Moreover, during plant disease, fungi produce different components that are essential to complete the infection process (enzymes, toxins, etc) and are named “pathogenicity factors”. Two dimensional gel electrophoresis (2-DE) has been widely used to study the proteome of a good number of microorganisms. However, few of them have been carried out to study the proteome of phytopathogenic fungi, mainly due to the difficulty of obtaining fungal protein extracts and/or the lack of available fungal protein databases. This review shows the strategies that can be addressed to overcome those problems and how different approaches have resulted to be useful in (i) obtaining the proteomic maps of several phytopathogenic fungi, (ii) determining fungal proteins involved in the formation of infective structures and (iii) studying the production of extracellular proteins or pathogenicity factors in phytopathogenic fungi. Fungal proteomic is still at an early stage, but recent reports show that it is a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research.

Global characterisation of proteins and the comparison between proteomes is providing new insights into general biological structures and processes, as well as between physiological and pathological conditions. During both embryonic development and adult life, brain cavities and ventricles are filled with a complex protein-rich fluid, the cerebrospinal fluid (CSF). In the last few years, it has been suggested that adult CSF has a key function as a fluid pathway for delivering diffusible signals to the brain, thus affecting the behaviour of specific brain parenchyma cells, particularly the putative neural stem cells. Accordingly, some pathologies that affect the central nervous system (CNS), such as neurodegenerative diseases and/or neurological disorders, are reflected in CSF protein content, either as a cause or as a consequence. Adult CSF is considered to be the successor of embryonic CSF (E-CSF). Most of the proteins contained within E-CSF, as analysed in avians (chick) and mammals (rat and human), are also detected in adult humans, suggesting that some of their roles in CNS cell behaviour are similar. Despite the heterogeneity of the currently available data, which is due to technical differences in the proteomic analyses, the use of different model systems and the great variety of sample sources, it is increasingly clear that this fluid regulates the behaviour of neural stem cells throughout development and during adult life, thus suggesting a possible approach to brain cell therapies. This review focuses on current data regarding CSF proteomes in relation to CSF functions in both physiological and pathological conditions, as well as on CSF manufacture.

The bee venom is used for treating a wide variety of conditions from acute tendonitis to chronic back pain to rheumatoid arthritis (RA). The major treatment is gene therapy or recombinant DNA vaccines involved in targeting multiple antigenic components to direct and empower the immune system to protect the host from infection. Limitations of therapy for the treatment of patients suffering from various adverse reaction and contraindications are always experienced. Antigenic epitopes on melittin protein of Apis dorsata are important determinants of protection against rheumatoid arthritis. As our knowledge of the immune responses to a protein antigen progressed, it became clear that the whole protein is not necessary for raising the immune response, but small segments as 4-AILKVLSTGLPALIS-18 of protein called the antigenic determinants or the epitopes, are sufficient for eliciting the desired immune response. Immunization cassettes should be capable of immunizing broad immunity against both humoral and cellular epitopes, thus giving vaccines the maximum ability to deal with A. dorsata immune escape against rheumatoid arthritis.

We have produced a safe and convenient “fluorescence imager” to observe the protein bands and spots in the polyacrylamide gel stained by the fluorescent stain. Coomassie brilliant blue (CBB) and the argentation or silver stain method have been used usually for this staining. The staining of the gel using a fluorescent stain, such as SYPRO Ruby was developed recently. This fluorescent stain has more advantages compared to the CBB and silver stain method. The most common fluorescent stain used for observation of the protein bands analyzed by polyacrylamide gel electrophoresis is SYPRO Ruby. To detect the SYPRO Ruby stained bands, an ultraviolet (UV) transilluminator or a large and expensive laser scanner is required. A UV transilluminator is usually used because fluorescent stain scanners are cumbersome and expensive for this purpose. Although UV light is harmful to eyes and skin, it has been used usually to identify and excise the target protein bands and spots in the gel. In addition to the need for, protection of the users, UV light damages the proteins. We have produced a convenient and safer fluorescent stained gel imager to avoid the above problems. The sensitivity of our device is as high as that of the laser scanner, the excitation light is not harmful to the human body and does not damage proteins. Additionally it is possible to produce various sizes of devices required for this purpose.