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PubMed2002

Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains.

Proteomics has traditionally used the separating power of two-dimensional electrophoresis for the quantitative analysis of protein amounts in complex extracts. However, the limitations of this approach in terms of throughput and analyzable protein range have elicited the development of other proteomics approaches, based either on peptide separations instead of protein separations, or based on direct protein recognition and selection on dedicated arrays (protein chips). These recent methods seem very promising, and probably look more promising than they will ultimately be, just because their weaknesses are not fully characterized yet. The purpose of this paper is thus to highlight the strengths and weaknesses of all the proteomics approaches proposed to date and to try to deduce the respective niches in proteomics that these approaches will have in the future.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2002

Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two-dimensional electrophoresis.

Comprehensive analysis of complex proteomes requires prefractionation of samples prior to two-dimensional gel electrophoresis (2-DE). This study demonstrates the utility of using a high resolution sample prefractionation method and slightly overlapping narrow pH range two-dimensional gel electrophoresis to enhance quantitative comparisons of complex proteomes. A key feature of this strategy is to prefractionate samples into a few well-defined pools using microscale solution isoelectric focusing (nusol-IEF) prior to 2-DE protein analysis. Sample prefractionation is achieved using a series of tandem small volume chambers (500 nuL) separated by thin membranes containing immobilines at specific pH's. The resulting well-resolved fractionated samples are optimally separated on a series of slightly overlapping narrow pH range immobilized pH gradient (IPG) gels, which are approximately 0.1 pH units wider than the nusol-IEF fractionated pools. When nusol-IEF prefractionation was applied to proteome analyses of mouse serum, it resulted in the capacity to separate much higher protein loads on narrow pH range IPG gels while retaining good resolution and spot recovery. More importantly, the prefractionation of serum greatly enhanced the ability to detect low abundance proteins, because major interfering proteins were removed from most fractions. At least 6- to 30-fold higher protein loads were possible for nonalbumin fractions on narrow pH range IPG gels. The dynamic range of protein detection is substantially increased since higher protein loads can be applied to narrow pH range 2-DE gels, and duplicate gels can be stained with colloidal Coomassie and silver stains for quantitation of abundant and minor proteins, respectively. Finally, the ability to effectively fractionate complex proteomes into very narrow ranges (< 0.5 pH units) strongly suggests that nusol-IEF could be used to prefractionate complex samples for subsequent direct analysis by liquid chromatography-tandem mass spectrometry methods as an alternative to using overlapping narrow pH range 2-DE gels.

Animals↗

PubMed2001

Global profiling of gene expression in cancer using genomics and proteomics.

Global profiling of gene expression at the genomic level using DNA microarrays and at the protein level using a variety of technologies have followed separate paths. Studies of gene expression for several types of tumors, using DNA microarrays, have been published recently that are informative with respect to delineating distinct patterns of gene expression among subsets of related tumors. Proteomics-based profiling uniquely allows delineation of global changes in protein expression patterns resulting from transcriptional and post-transcriptional control, post-translational modifications and shifts in proteins between different cellular compartments. Some of the current technologies for proteome profiling and the application of proteomics to the analysis of tumor tissues are reviewed. Given that comprehensive expression profiles obtained using genomics and proteomics are highly complementary, a combined approach to profiling may well uncover expression patterns that could not be predicted using a single approach.

Gene Expression Profiling↗

PubMed2002

[Differential proteomic analysis of human lung adenocarcinoma cell line A-549 and of normal cell line HBE].

To explore the differential proteomic expressions between human lung adenocarcinoma cell line A-549 and normal cell line HBE, a series of methods, including immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis, silver staining, PDQuest 2-DE software analysis, peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database searching, were used to separate and identify the differential proteomic expressions between A-549 and HBE. The results showed that the good 2-DE pattern including high resolution and reproducibility was obtained. After silver staining, the 2-DE image analysis by PDQuest 2-DE software detected average (890 +/- 38) spots in A-549, and (757 +/- 27) spots in HBE. The average positional deviation of the matched spots between A-549 and HBE 2-DE maps was (2.85 +/- 0.48) mm in IEF direction, and (2.69 +/- 0.37) mm in SDS-PAGE direction. The differential proteomic expression analysis found that there were 535 matched spots between A-549 and HBE 2-DE maps, 355 spots that were not matched in A-549, 222 spots that were not matched in HBE. 18 differential spots (8 spots in A-549 and 10 spots in HBE) were cut off from silver staining gel at random, digested in gel with TPCK-trypsin, measured with MALDI-TOF-MS and searched in the SWISS-PROT database with PeptIdent software. 18 protein were preliminarily identified. These proteins were related to cell signal transduction, cell metabolism, proliferation and differentiation etc. There was a significant difference at protein level between human lung adenocarcinoma cell line A-549 and normal cell line HBE. It suggests that the differential expression analysis of proteomes may be useful to further study of the related proteins and the molecular markers of lung adenocarcinoma.

Adenocarcinoma↗

PubMed2002

[Proteomics in clinical enzymology: polymorphism of creatine kinase and alkaline phosphatase].

INTRODUCTION: The clinical proteomics is dedicated to the use of the proteomics in the clinical laboratory science. AIMS: Examples for the importance of clinical proteomics are provided by studies on the polymorphisms of creatine kinase and alkaline phosphatase enzymes. METHODS: The different (immunoinhibition, electrophoretic, immunochemistry) assays for determination of a cardiac marker (creatine kinase 2) are compared in hospital patients. Isoform analysis of alkaline phosphatase is performed in benign, transient hyperphosphatasemia. RESULTS: The author reviews recent knowledge on polymorphism of creatine kinase (isoenzymes, isoforms, macro types), and alkaline phosphatase (isoenzymes, their cancer related variants, isoforms). The origin of falsely high cardiac marker (creatine kinase 2) determined by the immunoinhibition assay could be related to the presence of macro creatine kinase (both types), creatine kinase isoenzyme 1, and their mixtures. After reviewing the recent findings on the syndrome of benign, transient hyperphosphatasemia, a case of a 1-year-old Hungarian boy with this syndrome is presented. CONCLUSIONS: The author points out that the appropriate use of clinical proteomics (the polymorphisms of creatine kinase and alkaline phosphatase enzymes) may improve the diagnostic efficiency of the clinical laboratory tests.

Alkaline Phosphatase↗

PubMed2000

Identifying cytotoxic T cell epitopes from genomic and proteomic information: "The human MHC project.".

Complete genomes of many species including pathogenic microorganisms are rapidly becoming available and with them the encoded proteins, or proteomes. Proteomes are extremely diverse and constitute unique imprints of the originating organisms allowing positive identification and accurate discrimination, even at the peptide level. It is not surprising that peptides are key targets of the immune system. It follows that proteomes can be translated into immunogens once it is known how the immune system generates and handles peptides. Recent advances have identified many of the basic principles involved. The single most selective event is that of peptide binding to MHC, making it particularly important to establish accurate descriptions and predictions of peptide binding for the most common MHC variants. These predictions should be integrated with those of other steps involved in antigen processing, as these become available. The ability to translate the accumulating primary sequence databases in terms of immune recognition should enable scientists and clinicians to analyze any protein of interest for the presence of potentially immunogenic epitopes. The computational tools to scan entire proteomes should also be developed, as this would enable a rational approach to vaccine development and immunotherapy. Thus, candidate vaccine epitopes might be predicted from the various microbial genome projects, tumor vaccine candidates from mRNA expression profiling of tumors ("transcriptomes") and auto-antigens from the human genome.

Antigen Presentation↗

PubMed2002

Proteomics of human breast ductal carcinoma in situ.

We report the first proteomic analysis of matched normal ductal/lobular units and ductal carcinoma in situ (DCIS) of the human breast. An understanding of the transition from normal epithelium to the first definable stage of cancer at the functional level of protein expression is hypothesized to contribute to improved detection, prevention, and treatment. Ten sets of two-dimensional gels were evaluated, containing either matched normal ductal/lobular units or DCIS from either whole tissue sections or up to 100,000 laser capture microdissected epithelial cells. Differential protein expression was confirmed by image analysis. Protein spots (315) were excised and subjected to mass spectrometry sequencing. Fifty-seven proteins were differentially expressed between normal ductal/lobular units and DCIS. Differences in overall protein expression levels and posttranslational processing were evident. Ten differentially expressed proteins were validated in independent DCIS specimens, and 14 of 15 proteomic trends from two-dimensional gel analyses were confirmed by standard immunohistochemical analysis using a limited independent tumor cohort. Many of the proteins identified were previously unconnected with breast cancer, including proteins regulating the intracellular trafficking of membranes, vesicles, cancer preventative agents, proteins, ions, and fatty acids. Other proteomic identifications related to cytoskeletal architecture, chaperone function, the microenvironment, apoptosis, and genomic instability. Proteomic analysis of DCIS revealed differential expression patterns distinct from previous nucleic acid-based studies and identified new facets of the earliest stage of breast cancer progression.

Breast Neoplasms↗

PubMed2003

[Application of proteomic approach for solid tumor marker discovery].

In the post-genome era, the major mission of biology is to understand the functions of genome. To well know the whole book of genes, the proteins coded by different genes have to be studied more deeply since they are the final representatives of the specific genes. Proteomics is the key issue in the bioscience research. Using the two dimensional electrophoresis technique, the total proteins of tumor cells or tissues can be profiled first by their molecular weight and isoelectric points, after that, the specific interested proteins can be identified via mass spectrometry analysis combining with database searching. Cancer proteomics is a main part of the proteomics, by which we can understand the protein profiles during the different stages of the tumorigenesis and it brought a new hope for discovery of the tumor-specific biomarkers. Recently using proteomic tools with the laser capture microdissection (LCM) technique, more achievements have been made and many promising candidates of tumor-markers have been identified even most of them have not affirmed yet. This article will give a brief review about that.

Biomarkers, Tumor↗

PubMed2003

The human pituitary proteome: the characterization of differentially expressed proteins in an adenoma compared to a control.

In order to clarify the basic molecular mechanisms that participate in the formation of human pituitary macroadenomas, this study, for the first time, describes the comparative proteomics between a pituitary adenoma tissue and a control tissue. A vertical, two-dimensional polyacrylamide gel electrophoresis system and PDQuest image analysis software were used to provide a high level of between-gel reproducibility and electrophoretic separation to accurately locate each differentially expressed protein. Mass spectrometry (MALDI-TOF and LC-ESI-Q-IT) and protein databases were used to characterize each differentially expressed protein. A total of 137 differential gel spots (37 increased spot volumes, 39 decreased, 19 new and 42 lost) were found when we compared an adenoma proteome to a control proteome. Seventy-one spots (20 increased, 27 decreased, 13 new, 11 lost), representing 39 differentially regulated proteins, were identified. Five differentially regulated proteins (prolactin, cellular retinoic acid-binding protein II, G-protein beta subunit 3, secretagogin and calreticulin) were also validated with results from a comparative transcriptomics study of pituitary adenomas and controls. The functional characteristics of these differentially expressed proteins provide a differential proteomic profile between a pituitary adenoma and a control.

Adenoma↗

PubMedDate not supplied

Mass spectrometry-based proteomics and analyses of serum: a primer for the clinical investigator.

The vocabulary of proteomics and the swiftly-developing, technological nature of the field constitute substantial barriers to clinical investigators. In recent years, mass spectrometry has emerged as the most promising technique in this field. The purpose of this review is to introduce the field of mass spectrometry-based proteomics to clinical investigators, to explain many of the relevant terms, to introduce the equipment employed in this field, and to outline approaches to asking clinical questions using a proteomic approach. Examples of clinical applications of proteomic techniques are provided from the fields of cancer and vasculitis research, with an emphasis on a pattern recognition approach.

Blood Proteins↗

PubMedDate not supplied

[Micro-array based technologies to study the proteome: technological progress and applications].

Since these twenty last years, there is an increasing interest for large-scale analysis of biological function. In the field of transcriptome, the emergence of microarray-based technologies and the design of DNA biochips allow high-throughput studies of RNA expression in cell and tissue at a given moment. In the field of proteome, methods of reference are still the 2D electrophoresis followed by analysis with mass spectrometry. Technological progress makes it possible to apply microarray methods to proteomics study : they are protein biochips or protein arrays. Expression analysis of proteins in a cell or a tissue in simultaneous and highly parallel way give further information for large-scale studies of signaling pathway. Numerous applications of protein microarray-based assays are described in basic biological research and in medical research to identify diagnostic biomarkers of inflammatory and cancerous pathologies and to find out news drugs and new therapeutic targets. This review summarizes concrete applications of microarray-based technology in the field of proteome, describes fundamental technical stages in protein array development and highlights critical points which will be useful to improve this emerging proteomic method.

Protein Array Analysis↗

PubMed2004

AT1 receptor blockade alters metabolic, functional and structural proteins after reperfused myocardial infarction: detection using proteomics.

Angiotensin II (AngII) type 1 receptor (AT1R) blockers (ARBs) limit left ventricular (LV) dysfunction and necrosis after reperfused myocardial infarction (RMI) and proteomics can detect changes in protein levels after injury. We applied proteomics to detect changes in levels of specific protein in the ischemic zone (IZ) and non-ischemic zone (NIZ) of dog hearts after in vivo RMI (90 min of anterior ischemia; 120 min of reperfusion) and treatment with intravenous vehicle (control) and the ARBs valsartan or irbesartan (10 mg/kg) over 30 min before RMI. We also assessed LV function, infarction and apoptosis. Both ARBs limited the RMI-induced LV dysfunction, infarct size and apoptosis. Proteomics detected differential expression of 5 randomly selected proteins in the IZ compared to the NIZ after RMI: decrease in a subunit of ATP synthase isoform precursor (consistent with increased conversion to a subunit under metabolic stress), M chain creatine kinase (consistent with cellular damage) and ventricular myosin light chain-1 (consistent with structural damage and decreased contractility); and increase in NAD+ -isocitrate dehydrogenase (ICDH) and alpha subunit and ATP synthase D chain (mitochondrial, consistent with metabolic dysfunction). Importantly, changes in NAD+ -ICDH and ATP synthase D chain were reversed by ARB therapy. Thus, proteomics can detect regional changes in metabolic, contractile, and structural proteins after RMI and several of these proteins are favorably modified by ARBs, suggesting that they may be novel therapeutic targets.

ATP Synthetase Complexes↗

PubMed2004

The role of liquid chromatography in proteomics.

Proteomics represents a significant challenge to separation scientists because of the diversity and complexity of proteins and peptides present in biological systems. Mass spectrometry as the central enabling technology in proteomics allows detection and identification of thousands of proteins and peptides in a single experiment. Liquid chromatography is recognized as an indispensable tool in proteomics research since it provides high-speed, high-resolution and high-sensitivity separation of macromolecules. In addition, the unique features of chromatography enable the detection of low-abundance species such as post-translationally modified proteins. Components such as phosphorylated proteins are often present in complex mixtures at vanishingly small concentrations. New chromatographic methods are needed to solve these analytical challenges, which are clearly formidable, but not insurmountable. This review covers recent advances in liquid chromatography, as it has impacted the area of proteomics. The future prospects for emerging chromatographic technologies such as monolithic capillary columns, high temperature chromatography and capillary electrochromatography are discussed.

Chromatography, Liquid↗

PubMed2005

[Proteomics and diagnostic application].

Proteomics research possesses even greater potential than the achievements of genomics research. Clinical tests which more accurately identify pathology are needed. Clinical tests are also classified in terms of the metabolome. On the other hand, the techniques of proteomics research appear to be useful for developing diagnostic systems, not just searching for biomarkers. 2DG and the mass spectrometer are representative proteomics tools, and are now being used in blood tests. It is possible that systemic manifestations will come to be understood on the basis of the data obtained from them, and that they will be used for a variety of screening tests. Use of one of them, SELDI, is particularly interesting. High-throughput protein expression techniques, for example, phage display and the IVV method, are becoming useful for producing innovative high-affinity diagnostic antibodies. Protein chips that use ELISA and fluorescence for detection can be regarded as simultaneous multi-item tests. Up until now there have been chips with several spots, but it should soon become possible to make high-density chips in addition. Array devices for SPR, a new principle for viewing protein-protein interactions, have already been developed. Although they may not be very sensitive, they are useful in antibody tests because they make it possible to measure affinity. Simultaneously using different types of detection systems on the thin gold film of the SPR array would make possible a wider range of tests than ever before. Moreover, enormous chip data processing has the potential to create a new concept in profiling test methodology. Proteomics research such as that described above has the potential to give rise to new concepts in the field of diagnosis.

Biomarkers↗

PubMed2004

[Establishment of 2-dE map of human low differentiation nasopharyngeal carcinoma cell line CNE-2 proteome].

OBJECTIVE: To establish a two-dimensional polyacrylamide gel electrophoresis (2-DE) map of human low differentiation nasopharyngeal carcinoma (NPC) cell line CNE-2 proteome. METHODS: Immobilized pH gradient 2-DE was applied to separate the total proteins of CNE-2 cells, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching were used to indentify proteins. RESULTS: A good 2-DE pattern with high resolution and reproducibility was obtained. Seventy-eight protein spots were incised from sliver staining gel and digested in gel by trypisin. Of them 77 maps of peptide mass fingerprints (PMF) were obtained and 48 proteins were identified. CONCLUSION: A reference 2-DE map of CNE-2 proteome has been established and the data coupled with similar proteome analysis of other NPC cell lines will expand the human nasopharyngeal carcinoma proteome database.

Amino Acid Sequence↗

PubMedDate not supplied

[Contribution of proteomic analysis to study cardiovascular physiopathology].

Proteomic is an innovative approach for determining without any priori the coordinated changes in protein levels contained in a biological sample. This technique now constitutes a powerful tool for revealing, in particular by differential analysis, biological responses and regulation of proteins. This field is becoming essential in each domain for fundamental and clinical biology, mainly in human medicine. It leads to improve the understanding of complex physiopathological processes like those involved in human pathologies, but also for identifying new biomarkers in order to consider new diagnostic and prognostic approaches and original therapeutic targets. The different proteomic techniques, essentially bi-dimensional electrophoresis and mass spectrometry, which benefit from recent technological developments, are presented in this article. A literature review summarize data obtained from proteomic works performed to have better understanding of mechanisms involved in cardiovascular diseases, first cause of morbidity and mortality in the world. Proteomic analysis performed in this area of research are promising, because of the lack of knowledge in the molecular interactions and the complexity of mechanisms involved, and of the impact of environmental risk factors. This impact is being generally only detectable at protein level. These works are only at the beginning and these approaches applied in cardiovascular field will meet a large success in the immediate future and should be promising.

Cardiovascular Diseases↗

PubMed2005

Differential proteomic analysis in the study of prokaryotes stress resistance.

Proteomic technologies are powerful tools to study the physiological response of bacteria to various environmental stress conditions. Complex protein toolkits coordinated by regulatory networks have evolved to accommodate bacterial survival under several stressing conditions, such as varying temperature or pH, and changes in nutrient availability. This manuscript provides an overview of the major achievements obtained by 2D gel based and non-gel based proteomic approaches for the study of adaptive response in bacteria. As an example, a study carried out on Streptococcus thermophilus response to various stresses is here reported. In this investigation, we used combined proteomic strategies to define proteomic maps useful to highlight protein profile variations following environmental challenges.

Adaptation, Physiological↗

PubMed2006

Present status of clinical proteomic analysis for the early detection and determination of therapeutic strategy in lung cancer.

Although human DNA sequences have already been successfully decoded, there is still much we do not understand about the pathogenesis of human diseases. Proteomic analysis is comprehensive analysis of proteins, and there is a high probability that recent advances in this field will enable its application to individualized therapy based on thorough understanding of the pathogenesis of human diseases. Two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometry (MS) are indispensable tools for proteomic analysis, and these technologies enable discovery of tiny changes of protein expression associated with pathogenesis. In this review we introduce the present status of proteomic analysis for cancer-related proteins, and especially lung cancer-related proteins in the attempt to discover new biomarkers for early detection and determine novel target-molecules for treatment. We believe that proteomic analysis will provide crucial information for diagnosis and treatment.

Adenocarcinoma↗