Current Proteomics - Volume 9, Issue 1, 2012

Proteomics is defined as the parallel separation, identification and quantification of all proteins present in tissue, cell cultures, subcellular fractions or biological samples. It is often associated with the analysis of protein properties such as e.g. posttranslational modifications and interactions. Since the mid-1990's proteomic techniques have undergone major improvements leading to higher resolution in the separation of proteins, improved sensitivity and sample throughput combined with supercomputer based data processing in order to handle the vast amount of generated data. To fully benefit from the power of proteomics, particularly separation techniques for the recovery of all proteins in a given sample as well as quantification of proteins which can be present at an extended dynamic range had to be improved. Historically, proteomics employed 2D gel electrophoresis where individual protein spots were identified by subsequent MALDI-TOF mass spectrometry. While 2D gel electrophoresis is still widely used, rapid evolution of mass spectrometry based techniques combined with improved labelling techniques and protein microarrays has enabled quick and ready access to proteomes and changes in proteomes providing new insights into the biology of cells. It is foreseeable that proteomic techniques will further advance and will in future provide an important discipline towards unravelling important question such as the emergence of cancer, numerous diseases and the response of biological systems to environmental signals. Current Proteomics will publish selected high-impact manuscripts in the field of proteomics which will enable the reader to remain up to date with this rapidly evolving young discipline.

Staphylococcus aureus, one of the major pathogenic bacteria, is associated with substantial morbidity and mortality. The disease burden of staphylococcal infections is significant, which is primarily attributed to its adaptability and resistance to environmental stresses. S. aureus has the ability to develop multiple resistances to antimicrobial agents. These high resistances make pathogenicity of S. aureus one of the most complex mechanisms to understand and manage. Proteomic and bioinformatics approaches show great potential in exploring microbial adaptation strategies, ability to cause disease by pathogenic bacteria and the development of diagnostic tools. A summary of the latest developments in the application of ‘omics’ technologies to understand resistance mechanisms in S. aureus and their future role in antistaphylococcal vaccine and/or drug discovery is given here.

Nitric oxide is generated in almost all the tissues of the mammalian system however, under pathological conditions NO has damaging effects. Its increased concentration, under various neural diseases leads to cell damage through formation of highly reactive peroxynitrite. Cysteine proteinase inhibitors are of prime physiologic importance inside the cells, controlling the activities of lysosomal cysteine proteases. They prevent unwanted proteolysis and are involved in several neurodegenerative diseases. Our present study was designed to investigate the protective effect of curcumin and quercetin against NO induced damage of buffalo brain cystatin (BC) and to evaluate antinitrostative efficacy of curcumin and quercetin. Nitric oxide induced structural modifications were followed by fluorescence spectroscopy and functional inactivation by monitoring the inhibition of caseinolytic activity of papain. 50 μM sodium nitroprusside (SNP used to produced NO) caused time dependent inactivation of BC with complete functional loss precipitating at 150 min. Curcumin (50 μM) and quercetin (250 μM) opposed such loss in papain inhibitory activity of BC. Loss in tertiary structure of BC (fluorescence quenching) was observed on exposure to NO. Inhibition of functional and structural SNP mediated damage of BC by curcumin (50 μM) and Quercetin (250 μM) reaffirms their NO scavenging potency. By the results obtained, it was concluded that curcumin and quercetin were able to protect BC against structural and functional damage to a very considerable level. Thus, in view of this study, it can be elucidate that Curcumin and quercetin have a significant potential in the treatment of diseases caused by nitrogen free radicals and this potential must be further explored for the development of novel drugs.

Two-dimensional gel electrophoresis is a standard tool of proteomic analysis, consisting of first dimensional protein separation according to the respective protein's isoelectric point, and the second dimensional separation according to the protein's molecular weight. In these gels, “chain-like” protein spots of equal molecular weight but different isoelectric points are common observations. When identified by mass spectrometry, these chain-like protein spots are identified as the same protein entry in the databases, thus representing “protein species” in the proteomic literature. In this review, we will discuss the factors responsible for the occurrence of multiple protein species, which mostly depend on posttranslational modifications (PTMs) such as phosphorylation and glycosylation and others. Additionally, we will show how internet-based prediction tools can be used for comparing the theoretical models to the actual gel pattern. This may present useful tools for the validation of biomarkers in health and disease.

Cancer cells dramatically alter their metabolism in order to increase the production rate of intermediates required for nucleic and fatty acid biosynthesis in rapidly proliferating cells. While not well understood, dysregulation of oncogenes and tumour suppressors appears to result in the altered expression of specific isoforms of glycolysis proteins. A full understanding of glycolytic alterations in cancer through a systems biology approach requires tools to observe changes in the set of proteins that make up the glycolytic proteome. We propose that a targeted proteomics approach employing multiple reaction monitoring (MRM) is an excellent strategy to quantitatively monitor sets of proteins, such as those making up the glycolytic proteome. MRM is particularly well suited to proteins of glycolysis as they are of moderate to high abundance. Such systems-based efforts provide a means to understand the mechanisms for an altered glycolytic proteome in cancer, perhaps leading to novel drug targets and metabolic signatures for use in cancer prognosis.

By means of ELISA, confocal microscopy, FACS, 2-DE, ImageJ software, MALDI-ToF-MS, and PMF, in this study we report the differentially phosphoproteins expressed in untreated as well as in AZT-treated K562 cells. As evidenced by ELISA, and partly confirmed by confocal microscopy and flow cytometry analysis, an overall increase in Ophosphorylation was found in the AZT-treated samples. Additional analyses allowed to identify 17 spots representing 10 phosphoproteins differentially expressed, proteins which are involved in many significant biological functions such as mRNA processing, assembly and/or transport of ribosome, protein folding, energetic metabolism, cytoskeleton motility, growth control, hypoxia tolerance, allergic and stress responses. Five out of 10 phosphoproteins were up-regulated in K562 cells previously exposed to 20 μM AZT for 3 h (i.e. calreticulin, stathmin, triosephosphate isomerase, stressinduced- phosphoprotein-1, peptidyl-prolyl cis-trans isomerase A). On the contrary, the other five phosphoproteins were down-regulated (i.e. nucleophosmin, lactoylglutathione lyase, 3-hydroxyacyl-CoA dehydrogenase type-2, heterogeneous nuclear ribonucleoproteins A2-B1, alpha-enolase). The proteins identified in the present study represent the first clear report of differential phosphoproteins expression upon AZT treatment of human chronic myeloid leukemia (K562) cells. Therefore, this type of proteomic analysis could be envisaged as a further tool useful for monitoring response(s) to the drug, like the toxic side effects observed in HIV-infected patients under AZT therapy.

Many normal physiological and disease-related pathophysiological processes are regulated by the interactions of complex signaling networks of pro- and anti-inflammatory markers, growth factors, soluble receptors and extracellular matrix proteins, as well as other cell–cell signaling proteins, which we define collectively as cytokines. Because multiple cell–cell signaling factors may be involved in a single biological process, detection of expression of multiple cytokines is essential to unraveling the mechanisms and effects of many disease processes. Cytokine antibody arrays have been developed to meet this growing demand for multiplexed protein detection. In particular, discovery and validation of diseaserelated protein biomarkers require high-throughput detection of many proteins simultaneously. This review will address the complexity of cytokine biology, discuss current and future antibody array technologies and explore their applications in cytokine biomarker discovery and validation for variety of human diseases. Specific examples of these applications will be presented, including the search for cytokine biomarkers related to neurological and neurodegenerative diseases (such as autism and Alzheimer’s), immunological disorders (including asthma), and various cancers.