Prostate cancer and the genomic revolution: Advances using microarray analyses

The emerging technology of microarray analysis allows the establishment of molecular portraits of prostate cancer and the discovery of novel genes involved in the carcinogenesis process. Many novel genes have already been identified using this technique, and functional analyses of these genes are currently being tested. The combination of microarray analysis with other recently developed high-throughput techniques, such as proteomics, tissue arrays, and gene promoter-methylation, especially using tissue microdissection methods, will provide us with more comprehensive insights into how prostate cancer develops and responds to gene-targeted therapies. Animal models of prostate cancer are being characterized by high throughput techniques to better define the similarities and differences between those models and the human disease, and to determine whether particular models may be useful for specific targeted therapies in pre-clinical studies. Although profiling of mRNA expression provides important information of gene expression, the development of proteomic technologies will allow for an even more precise global insight into cellular signaling and structural alterations during prostate carcinogenesis. Not only will the "omic" revolution change basic science, but it will lead to a new era of molecular medicine.     

Current knowledge about the functional roles of phosphorylative changes of membrane proteins in normal and diseased red cells

With the advent of proteomic techniques the number of known post-translational modifications (PTMs) affecting red cell membrane proteins is rapidly growing but the understanding of their role under physiological and pathological conditions is incompletely established. The wide range of hereditary diseases affecting different red cell membrane functions and the membrane modifications induced by malaria parasite intracellular growth represent a unique opportunity to study PTMs in response to variable cellular stresses. In the present review, some of the major areas of interest in red cell membrane research have been considered as modifications of erythrocyte deformability and maintenance of the surface area, membrane transport alterations, and removal of diseased and senescent red cells. In all mentioned research areas the functional roles of PTMs are prevalently restricted to the phosphorylative changes of the more abundant membrane proteins. The insufficient information about the PTMs occurring in a large majority of the red membrane proteins and the general lack of mass spectrometry data evidence the need of new comprehensive, proteomic approaches to improve the understanding of the red cell membrane physiology.     

Use of functional proteomics to investigate PKC epsilon-mediated cardioprotection: the signaling module hypothesis

The characterization of biological processes on the basis of alterations in the cellular proteins, or "proteomic" analysis, is a powerful approach that may be adopted to decipher the signaling mechanisms that underlie various pathophysiological conditions, such as ischemic heart disease. This review represents a prospectus for the implementation of proteomic analyses to delineate the myocardial intracellular signaling events that evoke cardioprotection against ischemic injury. In concert with this, the manifestation of a protective phenotype has recently been shown to involve dynamic modulation of protein kinase C-epsilon (PKC epsilon) signaling complexes (Ping P, Zhang J, Pierce WM Jr, and Bolli R. Circ Res 88: 59--62, 2001). Accordingly, "the signaling module hypothesis" is formulated as a plausible mechanism by which multipurpose stress-activated proteins and signaling kinases may function collectively to facilitate the genesis of cardioprotection.     

Comparative proteomic analysis of cancerous and adjacent normal lung tissues

Lung cancer is the leading cause of cancer-related mortality in industrialized countries. Unfortunately, most lung cancers are found too late for a cure, therefore early detection and treatment is very important. We have applied proteomic analysis by using two-dimensional gel electrophoresis and peptide mass fingerprinting techniques for examination of cancerous and adjacent non-cancerous lung tissues from the same patient. The aim of the study was to find proteins, which could be used as biomarkers for diagnosis and monitoring of this disease. Indeed, we found differences in expression of several proteins, related to various cellular activities, such as, chaperoning (e,g. GRP96, GRP78, HSP27), metabolism and oxidation stress (e.g. L-fucose, GST), cytoskeleton (e.g., tubulin beta 2/3, beta actin), cell adhesion (e.g. annexin A5/3), binding proteins (e.g. 14-3-3 theta) and signal transduction. These changes may be important for progression of carcinogenesis; they may be used as the molecular-support for future diagnostic markers.     

Proteomic insights into ubiquitin and ubiquitin-like proteins

The dynamic and specific modification of cellular proteins by members of the ubiquitin protein family is a vital regulatory mechanism that lies at the heart of almost all biological processes. Because of both their pervasive and complex nature, these regulatory pathways have been the target of many recent proteomic studies. Such works have provided numerous insights. Through the use of various mass spectrometry techniques, affinity purification methods, and/or chemical probes, large lists have begun to be compiled for the multitude of substrates, interacting partners, and enzymatic components of these regulatory circuits. Furthermore, similar tools have provided many insights into functional aspects such as their mechanisms of substrate specificity and enzymatic activity. This review provides a summary of these recent proteomic works, along with comments on future directions of the field.     

Atomic force microscopy: A powerful molecular toolkit in nanoproteomics

Analysing microbial cell surface proteins is a challenging task in current microbial proteomic research, which has major implications for drug design, vaccine development, and microbial monitoring. In this context, atomic force microscopy (AFM) has recently emerged has a powerful characterization platform, providing valuable insights into the surface proteome of microbial cells. The aim of this article is to show how advanced AFM techniques, that all have in common functionalization of the AFM tip with specific molecules, can be used to answer pertinent questions related to surface‐associated proteins, such as what is their spatial arrangement on the cell surface, and what are the forces driving their interaction with the environment?     

Proteomic shifts in embryonic stem cells with gene dose modifications suggest the presence of balancer proteins in protein regulatory networks

Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of "balancer" proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the "elasticity" of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions.     

Proteomics: addressing the challenges of multiple myeloma

Multiple myeloma (MM) is a malignancy of terminally differentiated B-lymphocytes that accounts for ~13% of all hematologic cancers. Despite a wealth of knowledge describing the molecular biology of MM as well as significant advances in therapeutics, this disease remains incurable. Since proteins govern the cellular structure and biological function, a wide selection of proteomic approaches holds great promise for increasing our understanding of this disease, such as by investigating the dynamic nature of protein expression, cellular and subcellular distribution, post-translational modifications, and interactions at both the cellular and subcellular levels. The aims of this review are to introduce the available and emerging proteomic technologies that have potential applications in the study of MM and to highlight the current status of proteomic studies of MM. To date, although there have been a limited number of proteomic studies in MM, those performed have provided valuable information with regard to MM diagnosis and therapy. The potential future application of proteomic technologies is expected to provide new avenues in MM diagnostics, individualized therapy design and therapy response surveillance for the clinician.     

Features and applications of blue-native and clear-native electrophoresis

1-D native electrophoresis is used for the separation of individual proteins, protein complexes, and supercomplexes. Stable and labile protein-protein interactions can be identified depending on detergent and buffer conditions. 1-D native gels are immediately applicable for in-gel detection of fluorescent-labeled proteins and for in-gel catalytic activity assays. 1-D native gels and blots are used to determine native mass and oligomeric state of membrane proteins. Protein extracts from 1-D native gels are used for generation of antibodies, for proteomic work, and for advanced structural investigations. 2-D separation of subunits of protein complexes by SDS-PAGE is mostly used for immunological and proteomic studies. Following the discussion of these general features, specific applications of native electrophoresis techniques in various research fields are highlighted: immunological and receptor studies, biogenesis and assembly of membrane protein complexes, protein import into organelles, dynamics of proteasomes, proteome and subproteome investigations, the identification and quantification of mitochondrial alterations in apoptosis, carcinogenesis, and neurodegenerative disorders like Parkinson's disease, Alzheimer's disease, and the vast variety of mitochondrial encephalomyopathies.     

Proteome profile changes during transdifferentiation of NRP-152 rat prostatic basal epithelial cells

NRP-152 is an androgen responsive, non-tumorigenic cell line, which shows basal epithelial cell characteristics under normal growth conditions. It has been noted that NRP-152 undergoes morphological and cytoskeletal changes toward its luminal counterpart NRP-154 when it is grown under growth restrictive conditions. We have extensively investigated the details of protein change of NRP-152 during transdifferentiation using proteomic techniques. NRP-152 cells were cultured under normal and growth restrictive media conditions for 3, 5 days. NRP-154 cells were normally cultured. Protein samples were submitted to 2D gel electrophoresis and silver stained. Protein patterns on the gels were comparatively analysed using Melanie III software. Protein spots exhibiting significant changes in NRP-152 cells during the time course were excised and subjected to in-gel tryptic digestion. After 6 days of growth restrictive conditions in NRP-152, the cells were morphologically changed resembling luminal phenotype. Of the 35 protein spots that were up-reglated, 20 proteins from 21 spots were identified by peptide mass fingerprinting and, of 21 proteins spots that were down-regulated, 10 proteins from 12 spots were identified as landmark proteins. Our study confirmed that basal NRP-152 cells were proportionally transdifferentiated into luminal featuring cells according to the duration of growth restrictive culture conditions. This suggests that human prostatic basal epithelial cells may be changed into luminal cells under certain conditions. Proteomic approach enabled us to identify 30 proteins involved in this differentiation with a single experiment. These proteins will be subjected to further functional studies to evaluate their possible roles related to cellular differentiation. These data strongly support that proteomics is a very powerful approach for studying physiologic and pathologic cellular changes such as differentiation and carcinogenesis.     

Comprehensive proteomic profiling of adult Angiostrongylus costaricensis, a human parasitic nematode

Angiostrongylus costaricensis is a nematode helminth that causes an intestinal acute inflammatory process known as abdominal angiostrongyliasis, which is a poorly understood human disease occurring in Latin America. Our aim was to study the proteomic profiles of adult parasites focusing on immunogenic proteins. Total cellular extracts from both genders showed similar 2-DE profiles, with 60% of all protein spots focused between pH 5-7 and presenting molecular masses from 20.1 to 66 kDa. A total of 53 different dominant proteins were identified in our dataset and were mainly associated with the following over-represented Gene Ontology Biological Process terms: "macromolecule metabolic process", "developmental process", "response to stress", and "biological regulation". Female and male immunoblots showed similar patterns of reactive proteins. Immunoreactive spots identified by MALDI-PSD were found to represent heat shock proteins, a putative abnormal DAuer Formation family member, and galectins. To date, very few biochemical analyses have focused on the nematode Angiostrongylus costaricensis. As such, our results contribute to a better understanding of its biology and the mechanisms underlying the host-parasite relationship associated with this species. Moreover, our findings represent a first step in the search for candidate proteins for diagnostic assays and the treatment of this parasitic infection.     

Toxicology and genetic toxicology in the new era of "toxicogenomics": impact of "-omics" technologies.

The unprecedented advances in molecular biology during the last two decades have resulted in a dramatic increase in knowledge about gene structure and function, an immense database of genetic sequence information, and an impressive set of efficient new technologies for monitoring genetic sequences, genetic variation, and global functional gene expression. These advances have led to a new sub-discipline of toxicology: "toxicogenomics". We define toxicogenomics as "the study of the relationship between the structure and activity of the genome (the cellular complement of genes) and the adverse biological effects of exogenous agents". This broad definition encompasses most of the variations in the current usage of this term, and in its broadest sense includes studies of the cellular products controlled by the genome (messenger RNAs, proteins, metabolites, etc.). The new "global" methods of measuring families of cellular molecules, such as RNA, proteins, and intermediary metabolites have been termed "-omic" technologies, based on their ability to characterize all, or most, members of a family of molecules in a single analysis. With these new tools, we can now obtain complete assessments of the functional activity of biochemical pathways, and of the structural genetic (sequence) differences among individuals and species, that were previously unattainable. These powerful new methods of high-throughput and multi-endpoint analysis include gene expression arrays that will soon permit the simultaneous measurement of the expression of all human genes on a single "chip". Likewise, there are powerful new methods for protein analysis (proteomics: the study of the complement of proteins in the cell) and for analysis of cellular small molecules (metabonomics: the study of the cellular metabolites formed and degraded under genetic control). This will likely be extended in the near future to other important classes of biomolecules such as lipids, carbohydrates, etc. These assays provide a general capability for global assessment of many classes of cellular molecules, providing new approaches to assessing functional cellular alterations. These new methods have already facilitated significant advances in our understanding of the molecular responses to cell and tissue damage, and of perturbations in functional cellular systems.As a result of this rapidly changing scientific environment, regulatory and industrial toxicology practice is poised to undergo dramatic change during the next decade. These advances present exciting opportunities for improved methods of identifying and evaluating potential human and environmental toxicants, and of monitoring the effects of exposures to these toxicants. These advances also present distinct challenges. For example, the significance of specific changes and the performance characteristics of new methods must be fully understood to avoid misinterpretation of data that could lead to inappropriate conclusions about the toxicity of a chemical or a mechanism of action. We discuss the likely impact of these advances on the fields of general and genetic toxicology, and risk assessment. We anticipate that these new technologies will (1) lead to new families of biomarkers that permit characterization and efficient monitoring of cellular perturbations, (2) provide an increased understanding of the influence of genetic variation on toxicological outcomes, and (3) allow definition of environmental causes of genetic alterations and their relationship to human disease. The broad application of these new approaches will likely erase the current distinctions among the fields of toxicology, pathology, genetic toxicology, and molecular genetics. Instead, a new integrated approach will likely emerge that involves a comprehensive understanding of genetic control of cellular functions, and of cellular responses to alterations in normal molecular structure and function.     

Proteomic analysis of liver tissue from HBx‐transgenic mice at early stages of hepatocarcinogenesis

The hepatitis B virus X‐protein (HBx), a multifunctional viral regulator, participates in the viral life cycle and in the development of hepatocellular carcinoma (HCC). We previously reported a high incidence of HCC in transgenic mice expressing HBx. In this study, proteomic analysis was performed to identify proteins that may be involved in hepatocarcinogenesis and/or that could be utilized as early detection biomarkers for HCC. Proteins from the liver tissue of HBx‐transgenic mice at early stages of carcinogenesis (dysplasia and hepatocellular adenoma) were separated by 2‐DE, and quantitative changes were analyzed. A total of 22 spots displaying significant quantitative changes were identified using LC‐MS/MS. In particular, several proteins involved in glucose and fatty acid metabolism, such as mitochondrial 3‐ketoacyl‐CoA thiolase, intestinal fatty acid‐binding protein 2 and cytoplasmic malate dehydrogenase, were differentially expressed, implying that significant metabolic alterations occurred during the early stages of hepatocarcinogenesis. The results of this proteomic analysis provide insights into the mechanism of HBx‐mediated hepatocarcinogenesis. Additionally, this study identifies possible therapeutic targets for HCC diagnosis and novel drug development for treatment of the disease.     

Proteomics techniques for the development of flood tolerant crops

Proteomics is a useful analytical approach for investigating crop responses to stress. Recent remarkable advances in proteomic techniques allow for the identification of a wider range of proteins than was previously possible. The application of proteomic techniques to clarify the molecular mechanisms underlying crop responses to flooding stress may facilitate the development of flood tolerant crops. Flooding is an environmental stress found worldwide and may increase in frequency due to changes in global climate. Waterlogging resulting from flooding causes significant reductions in the growth and yield of several crops. Transient flooding displaces gases in soil pores and often causes hypoxia in plants grown on land with poor drainage. Changes in protein expression and post-translational modification of proteins occur as plants activate their defense system in response to flooding stress. In this review, we discuss the contributions that proteomic studies have made toward increasing our understanding of the well-organized cellular response to flooding in soybean and other crops. The biological relevance of the proteins identified using proteomic techniques in regard to crop stress tolerance will be discussed as well.     

Proteomic tools for cell biology

Acquisition of large bodies of genomic sequence is facilitating the use of global techniques to assay cellular function. DNA microarrays have enabled the measurement of global mRNA levels and are able to detect changes in gene expression between different cellular states. Since much of the regulation of physiolgical processes happens post-translationally, measuring only the mRNA levels gives an incomplete picture. Strategies to assay global expression, localization, or interaction of proteins fall into the emerging field of proteomics, with various combinations of techniques being utilized to separate and identify proteins. In this review, we will present a general overview of the currently available proteomic tools and then give examples of how these tools are being utilized to answer questions in cell biology.     

Advanced proteomic technologies for cancer biomarker discovery

Proteomic technologies have experienced major improvements in recent years. Such advances have facilitated the discovery of potential tumor markers with improved sensitivities and specificities for the diagnosis, prognosis and treatment monitoring of cancer patients. This review will focus on four state-of-the-art proteomic technologies, namely 2D difference gel electrophoresis, MALDI imaging mass spectrometry, electron transfer dissociation mass spectrometry and reverse-phase protein array. The major advancements these techniques have brought about and examples of their applications in cancer biomarker discovery will be presented in this review, so that readers can appreciate the immense progress in proteomic technologies from 1997 to 2008. Finally, a summary will be presented that discusses current hurdles faced by proteomic researchers, such as the wide dynamic range of protein abundance, standardization of protocols and validation of cancer biomarkers, and a 5-year view of potential solutions to such problems will be provided.     

Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues

Formalin-fixed and paraffin-embedded tissues represent the vast majority of archived tissue. Access to such tissue specimens via shotgun-based proteomic analyses may open new avenues for both prospective and retrospective translational research. In this study, we evaluate the effects of fixation time on antigen retrieval for the purposes of shotgun proteomics. For the first time, we demonstrate the capability of a capillary isotachophoresis (CITP)-based proteomic platform for the shotgun proteomic analysis of proteins recovered from FFPE tissues. In comparison to our previous studies utilizing capillary isoelectric focusing, the CITP-based analysis is more robust and increases proteome coverage. In this case, results from three FFPE liver tissues yield a total of 4098 distinct Swiss-Prot identifications at a 1% false-discovery rate. To judge the accuracy of these assignments, immunohistochemistry is performed on a panel of 17 commonly assayed proteins. These proteins span a wide range of protein abundances as inferred from relative quantitation via spectral counting. Among the panel were 4 proteins identified by a single peptide hit, including three clusters of differentiation (CD) markers: CD74, CD117, and CD45. Because single peptide hits are often regarded with skepticism, it is notable that all proteins tested by IHC stained positive.