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Proteomics is the study of total expressed proteins of genome in certain cell, tissue or organ. It is the frontier of the research in life science in the new century, and the core of functional genomics. Proteomics provides a new way to study ocular cell behaviors and the mechanisms of ocular disease. Here we reviewed the applications of proteomics in ophthalmology.
Proteomics has been widespreadly implanted into every field of life science and medicine as an important part of post-genomics era research. The development of theory and technology in proteomics has provided new ideas and research fields for cancer research. Proteomics can be used for not only elucidating the mechanism of carcinogenesis from the whole proteins of the tissue or cell but also seeking the biomarkers for diagnosis and therapy of cancer.
Separation and enrichment of organelles from complex biological mixtures are important for proteomic analysis. Two widely used current standard techniques to isolate individual organelles include differential and density-gradient centrifugation. Although these techniques have proven useful for processing small volumes of sample, multiple rounds of centrifugation are required when performing a large-scale purification. In this report, we have introduced a novel technique: continuous-flow ultracentrifugation using a sucrose gradient to separate, accumulate, and highly enrich bovine heart mitochondria in one step. To demonstrate the advantage of the technique, mitochondrial proteins from two different bovine hearts (3-8 mo and 18-30 mo old) were examined. For each age group, 100 g of bovine heart tissue were homogenized by a blending procedure. After removal of the nuclei, the entire remaining homogenate was loaded onto a proteomics continuous-flow ultracentrifuge to separate and enrich the organelles. Fractions were collected and mitochondria-enriched fractions were identified by Western blot analysis. To study the protein profile changes with aging in the mitochondrial proteome, the mitochondria-enriched fractions were applied to two-dimensional gel electrophoresis. The resulting two-dimensional PAGE gels were subsequently analyzed by image analysis software to identify proteins unique to each age group and proteins with at least twofold differences in protein expression. These proteins were then digested with trypsin and identified by mass spectrometer. Significant differences in the protein profiles of the two differently aged mitochondria preparations were found. The continuous-flow ultracentrifugation technique was demonstrated to be a powerful tool for separation and enrichment of organelles and their sub-types.
Proteomics, an important discipline in genomic era, is the integrated study of protein properties on a large scale. Application of proteomics researches in Alzheimer's disease (AD), which is one of the most common and complex neurodegeneration disorders, we can not only elucidate the essential issues of disease in protein level, but give a whole view in the pathological mechanisms identification, diagnosis and therapeutic targets discovery as well. The three aspects about the progresses in proteomics researches of AD were reviewed in this article.
Proteomic analysis combining two-dimentional electrophoresis (2DE) and mass spectrometry (MS) has the potential for a wide range of applications in biological and medical sciences, as protein screening in tissues obtained from healthy and diseased conditions can determine drug targets and diagnostic markers. Conventionally, amniotic fluid (AF) samples are routinely used for prenatal diagnosis of a wide range of fetal abnormalities. Proteomics have already been applied in the analysis of tissues from fetuses with Down's syndrome, in order to detect differences in their protein profile as compared to the normal profiles and to determine possible diagnostic tools. A detailed protein 2DE for the normal human AF has not been reported. In the present study, the 2D protein database of the normal human AF supernatant (AFS) was constructed. Ten AFS samples from women carrying normal fetuses were analysed by 2DE. A mean of 412 spots per gel were analyzed and protein identification was carried out by MALDI-MS and MALDI-MS-MS. A 2D protein map comprising of 136 different gene products was constructed. The majority of the identified proteins are regulatory proteins, enzymes, secreted proteins, carriers and immunoglobulins. Twelve hypothetical proteins were also included. The normal AFS proteome map is a valuable tool for the study of aberrant protein expression and the search for proteins as possible markers for the prediction of abnormal fetuses.
CONTEXT: Intra-amniotic infection (IAI) is commonly associated with preterm birth and adverse neonatal sequelae. Early diagnosis of IAI, however, has been hindered by insensitive or nonspecific tests. OBJECTIVE: To identify unique protein signatures in rhesus monkeys with experimental IAI, a proteomics-based analysis of amniotic fluid was used to develop diagnostic biomarkers for subclinical IAI in amniotic fluid and blood of women with preterm labor. DESIGN, SETTING, AND PARTICIPANTS: Surface-enhanced laser desorption-ionization/time-of-flight mass spectrometry, gel electrophoresis, and tandem mass spectrometry were used to characterize amniotic fluid peptides in 19 chronically instrumented pregnant rhesus monkeys before and after experimental IAI. Candidate biomarkers were determined by liquid chromatography-tandem mass spectrometry. Polyclonal antibodies were generated from synthetic peptides for validation of biomarkers of IAI. Amniotic fluid peptide profiles identified in experimental IAI were subsequently tested in a cohort of 33 women admitted to Seattle, Wash, hospitals between June 25, 1991, and June 30, 1997, with preterm delivery at 35 weeks or earlier associated with subclinical IAI (n = 11), preterm delivery at 35 weeks or earlier without IAI (n = 11), and preterm contractions with subsequent term delivery at later than 35 weeks (n = 11). MAIN OUTCOME MEASURES: Identification of peptide biomarkers for occult IAI. RESULTS: Protein expression profiles in amniotic fluid showed unique signatures of overexpression of polypeptides in the 3- to 5-kDa and 10- to 12-kDa molecular weight ranges in all animals after infection and in no animal prior to infection. In women, the 10- to 12-kDa signature was identified in all 11 patients with subclinical IAI, in 2 of 11 with preterm delivery without IAI, and in 0 of 11 with preterm labor and term delivery without infection (P<.001). Peptide fragment analysis of the diagnostic peak in amniotic fluid identified calgranulin B and a unique fragment of insulinlike growth factor binding protein 1, which were also expressed in maternal serum. Mapping of other amniotic fluid proteins differentially expressed in IAI identified several immunoregulators not previously described in amniotic fluid. CONCLUSIONS: This proteomics-based characterization of the differential expression of amniotic fluid proteins in IAI identified a distinct proteomic profile in an experimental primate chorioamnionitis model that detected subclinical IAI in a human cohort with preterm labor. These diagnostic protein expression signatures, complemented by immunodetection of specific biomarkers in amniotic fluid and in maternal serum, might have application in the early detection of IAI.
Two-dimensional (2-D) polyacrylamide gel electrophoresis has much to contribute to experimental analysis of the proteomes of microbial organisms, since this method separates most cellular proteins and allows synthesis rates to be determined quantitatively. Databases generated using 2-D gels can grow to be very large from even just a few experiments, since each sample provides the data for a field (or column) in the database for several hundreds to even thousands of records (or rows), each of which represents a single polypeptide species. The value of such databases for generating an encyclopedia of how each of the cell's proteins behave in different conditions (protein phenotypes) has been recognized for some time. The potential exists, however, to glean even more valuable information from such databases. Because the measurements of each protein are made in the context of all other proteins, a comprehensive glimpse of the cell's physiological state is theoretically achievable with each 2-D gel. By examining enough conditions (and 2-D gels), expression patterns of subsets of proteins (proteomic signatures) can be found that correlate with the cell's state. This type of information can provide a unique contribution to proteomic analysis, and should be a major focus of such analyses.
The urological malignancies, renal, bladder and prostate cancer, account for approximately 16% of all cancer cases. Unfortunately 5-year survival rates are relatively poor, largely a result of many cases not being diagnosed before the tumour has metastasised. There is a clear need for the identification of markers which will allow earlier detection of disease, and predict prognosis and response to therapy. In addition, they may be of use as therapeutic targets. Current advances in molecular biology are allowing the identification of a number of tumour-associated changes which could be of clinical use in the future. However, with the rapid technological advances being made in the field of proteomics, this approach could be integrated with genomics providing a complementary alternative, overcoming disparities between mRNA levels and protein production, and additionally allowing the identification of tumour-associated post-translational modifications. These approaches have already been used to identify novel genes and other cancer-related changes involved in the pathogenesis of urological malignancies. This review describes current progress in the genomic and proteomic study of urological malignancies, and highlights the potential of using proteomic technologies in the study of this group of diseases.
Until recently scientists studied genes or proteins one at a time. With improvements in technology, new tools have become available to study the complex interactions that occur in biological systems. Global studies are required to do this, and these will involve genomic and proteomic approaches. High-throughput methods are necessary in each case because the number of genes and proteins in even the simplest of organisms are immense. In the developmental phase of genomics, the emphasis was on the generation and assembly of large amounts of nucleic acid sequence data. Proteomics is currently in a phase of technological development and establishment, and demonstrating the capacity for high throughput is a major challenge. However, funding bodies (both in the public and private sector) are increasingly focused on the usefulness of this capacity. Here we review the current state of proteome research in terms of capacity and utility.
Microbes present special opportunities for proteomic analysis that are not yet available for other types of organisms, due mainly to the relative abundance of information on their genomes, their low levels of functional redundancy and their experimental tractability. They are also being used to develop and validate powerful new experimental approaches that surmount some important current limitations in this field. The review surveys the different proteomic procedures that are available and considers the advantages and disadvantages of different experimental strategies. The ways in which microbiologists - and others - can exploit proteomic data are also discussed.
Bodily fluids such as cerebrospinal fluid (CSF) and serum can be analysed at the time of presentation and throughout the course of the disease. Changes in the protein composition of CSF may be indicative of altered CNS protein expression pattern with a causative or diagnostic disease link. These findings can be strengthened through subsequent proteomic analysis of specific brain areas implicated in the pathology. New isolation strategies of clinically relevant cellular material such as laser capture microdissection, protein enrichment procedures and proteomic approaches to neuropeptide and neurotransmitter analysis give us the opportunity to map out complex cellular interaction at an unprecedented level of detail. In neurological disorders multiple underlying pathogenic mechanisms as well as an acute and a chronic CNS disease components may require a selective repertoire of molecular targets and biomarkers rather than an individual protein to better define a complex disease. The resulting proteome database bypasses many ambiguities of experimental models and may facilitate pre- and clinical development of more specific disease markers and new selective fast acting therapeutics.
In this review we examine the current state of analytical methods in proteomics. The conventional methodology using two-dimensional electrophoresis gels and mass spectrometry is discussed, with particular reference to the advantages and shortcomings thereof. Two recently published methods which offer an alternative approach are presented and discussed, with emphasis on how they can provide information not available via two-dimensional gel electrophoresis. These two methods are the isotope-coded affinity tags approach of Gygi et al. and the two-dimensional liquid chromatography-tandem mass spectrometry approach as presented by Link et al. We conclude that both of these new techniques represent significant advances in analytical methodology for proteome analysis. Furthermore, we believe that in the future biological research will continue to be enhanced by the continuation of such developments in proteomic analytical technology.
Advances in DNA sequencing and the near-term availability of whole genome sequences for several pharmaceutically relevant organisms promise to dramatically alter the breadth and scale of high-throughput proteomic studies. The substantial amount of literature is available in the public domain, demonstrate the potential of proteomics in the preclinical phases of pharmaceutical development. Over the next few years, it is anticipated that functional genomics and proteomics will have major impacts on the clinical phases of drug development. Expected benefits are earlier proof-of-concept studies in man and increased efficiency of clinical trials through the availability of biologically relevant markers for drug efficacy and safety.
A proteomic analysis was performed comparing normal rat soleus muscle to denervated soleus muscle at 0.5, 1, 2, 4, 6, 8 and 10 days post denervation. Muscle mass measurements demonstrated that the times of major mass changes occurred between 2 and 4 days post denervation. Proteomic analysis of the denervated soleus muscle during the atrophy process demonstrated statistically significant (at the p < 0.01 level) changes in 73 soleus proteins, including coordinated changes in select groups of proteins. Sequence analysis of ten differentially regulated proteins identified metabolic proteins, chaperone and contractile apparatus proteins. Together these data indicate that coordinated temporally regulated changes in the proteome occur during denervation-induced soleus muscle atrophy, including changes in muscle metabolism and contractile apparatus proteins.
The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. The aqueous and detergent phases were further fractionated by anion-exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy-dependent manner were characterized. All fractions were analyzed by two-dimensional (2-D) gel electrophoresis and identification numbers were given to 588 unique 2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and detergent phase 2-D gels. Fifty-one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.
A Proteome map of the bacterium Mycoplasma pneumoniae was constructed using two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry (MS). M. pneumoniae is a human pathogen with a known genome sequence of 816 kbp coding for only 688 open reading frames, and is therefore an ideal model system to explore the scope and limits of the current technology. The soluble protein content of this bacterium grown under standard laboratory conditions was separated by 1-D or 2-D gel electrophoresis applying various pH gradients, different acrylamide concentrations and buffer systems. Proteins were identified using liquid chromatography-electrospray ionization ion trap and matrix-assisted laser desorption/ionization-MS. Mass spectrometric protein identification was supported and controlled using N-terminal sequencing and immunological methods. So far, proteins from about 350 spots were characterized with MS by determining the molecular weights and partial sequences of their tryptic peptides. Comparing these experimental data with the DNA sequence-derived predictions it was possible to assign these 350 proteins to 224 genes. The importance of proteomics for genome analysis was shown by the identification of four proteins, not annotated in the original publication. Although the proteome map is still incomplete, it is already a useful reference for comparative analyses of M. pneumoniae cells grown under modified conditions.
The epidermal growth factor (EGF) is a member of the growth factor superfamily that can stimulate the proliferation of many types of cells. Overexpression of EGF receptor (EGFR) was observed in many types of cancer cells. Anti-EGFR antibodies or antisense nucleic acid sequences of EGFR can suppress the growth of hepatoma cells. In order to further investigate the proteome alterations associated with malignant growth of the human hepatoma cells and the influence of EGFR signal pathway on the cellular proteome, we have comparatively analyzed the proteomes of human hepatoma cells transfected with antisense EGFR sequence (cell strain JX-1) and its control cells (cell strain JX-0) by two-dimensional (2-D) gel electrophoresis and mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 40 protein spots showed significant expression changes in JX-1 cells compared to JX-0 cells. Three of them, including the tumor suppressor protein maspin, changed with tendency to the normal levels. Two protein spots were identified as HSP27 in the same gel, and one of them had a reduced level in JX-1 cells. The apparent alterations of HSP27 in expression level might be the results from their differential chemical modifications, suggesting the effect of dynamic post-translational modifications of proteins on the growth of hepatoma cells. Other proteins such as glutathione peroxidase (GPX-1) and 14-3-3-sigma also exhibited altered expression in JX-1 cells, and their functional implications are discussed.
Proteome analysis requires fast methods with high separation efficiencies in order to screen the various cell and tissue types for their proteome expression and monitor the effect of environmental conditions and time on this expression. The established two-dimensional gel electrophoresis (2-DE) is by far too slow for a consequential screening. Moreover, it is not precise enough to observe changes in protein concentrations. There are various approaches that promise faster, automated proteome analysis. This article concentrates on capillary (CT isoelectric focusing coupled to mass spectrometry (CIEF-MSn) and preparative IEF followed by size-exclusion chromatography, hyphenated with MS (PIEF-SEC-MS). These two approaches provide a similar separation pattern as the established 2-DE technique and therefore allow for the continued use of data based on this traditional approach. Their performances have been discussed and compared to 2-DE, evaluating 169 recent articles. Data on analysis time, automation, the detection limit, quantitation, peak capacity, mass and pI accuracy, as well as on the required sample amount are compared in a table.