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PubMed2005

Gel-based versus gel-free proteomics: a review.

With the sequencing of the genome of over 150 organisms, the field of biology has been revolutionised. Instead of studying one gene or protein at the time, it is now possible to study the effect of physiological or pathological changes on the expression of all genes or proteins in the organism. Proteomics aims at the simultaneous analysis of all proteins expressed by a cell, tissue or organism in a specific physiological condition. Because proteins are the effector molecules in all organisms, it is evident that changes in the physiological condition of an organism will be reflected by changes in protein expression and/or processing. Since the formulation of the concept of proteomics in the mid 90's proteomics has relied heavily on 2 dimensional gel electrophoresis (2DGE) for the separation and visualization of proteins. 2DGE, however, has a number of inherent drawbacks. 2DGE is costly, fairly insensitive to low copy proteins and cannot be used for the entire proteome. Therefore, over the years, several gel-free proteomics techniques have been developed to either fill the gaps left by 2DGE or to entirely abolish the gel based techniques. This review summarizes the most important gel-free and gel-based proteomics techniques and compares their advantages and drawbacks.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Proteolytic 18O-labeling strategies for quantitative proteomics.

A number of proteomic techniques have been developed to quantify proteins in biological systems. This review focuses on the quantitative proteomic technique known as "proteolytic 18O-labeling." This technique utilizes a protease and H(2)18O to produce labeled peptides, with subsequent chromatographic and mass spectrometric analysis to identify and quantify (relative) the proteins from which the peptides originated. The technique determines the ratio of individual protein's expression level between two samples relative to each other, and can be used to quantitatively examine protein expression (comparative proteomics) and post-translational modifications, and to study protein-protein interactions. The present review discusses various aspects of the 18O-labeling technique, including: its history, the advantages and disadvantages of the proteolytic 18O-labeling technique compared to other techniques, enzymatic considerations, the problem of variable incorporation of 18O atoms into peptides with a discussion on recent advancements of the technique to overcome it, computational tools to interpret the data, and a review of the biological applications.

Gene Expression Profiling↗
PubMed2006

Disulfide proteome yields a detailed understanding of redox regulations: a model study of thioredoxin-linked reactions in seed germination.

Accumulating evidence suggests that redox regulations play important roles in a broad spectrum of biological processes. Recently, Yano et al. developed a disulfide proteome technique that comprehensively visualizes redox change in proteins. In this paper, using the disulfide proteome, we examined rice bran and identified fragments of embryo-specific protein and dienelactone hydrolase as putative targets of thioredoxin. Also, monitoring of the endogenous and recombinant effects of thioredoxin on rice bran proteins and supporting in vivo observations propose a mechanism of redox regulation in seed germination, in which thioredoxin activates cysteine protease with a concurrent unfolding of its substrate, the embryo-specific protein. Our findings suggest that thioredoxin controls the lifetime of specific proteins effectively by regulating the redox reactions coordinately. The model study demonstrates that the disulfide proteome technique is useful not only for identifying targets of thioredoxin, but also for clarify the detailed mechanism of redox regulation.

Amino Acid Sequence↗
PubMed2005

Proteomic analysis of kidney in fluoride-treated rat.

The recent development of proteomic techniques has enabled investigators to directly examine the population of proteins present in biological systems. We first report here the proteomic changes of renal protein induced by fluoride. To investigate molecular mechanisms of renal injury induced by fluoride, proteins were isolated from rat kidney and profiled by two-dimensional gel electrophoresis (2DE). With the analysis of Image-Master 2D Elite software, 141 up-regulated and eight down-regulated protein spots in 2DE gels of fluoride-treated group were gained by comparison to the control group, 13 of which were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The identified proteins are mainly related with cell proliferation, metabolism and oxidative stress, and provide a valuable clue to explore the mechanism of renal fluorosis. This study also shows that the proteomic techniques were powerful in fluoride toxical field.

Animals↗
PubMed2006

HPLC techniques for proteomics analysis--a short overview of latest developments.

Due to the complex nature of the proteome, instrumentation and methods development for sample cleanup, fractionation, preconcentration, chromatographic separation and detection becomes urgent for the identification of peptides and proteins. Newly developed techniques and equipment for separation and detection, such as nano-HPLC and multidimensional HPLC for protein and peptide separation, enabled proteomics to experience dynamic growth during the past few years. In any proteomic analysis the most important and sometimes most difficult task is the separation of the complex mixture of proteins or peptides. This review describes some aspects and limitations of HPLC, both multidimensional and one-dimensional, in proteomics research without attempting to discuss all available HPLC methods, which would need far more space than available here.

Animals↗
PubMed2002

Proteomics in drug discovery.

Drug discovery is a prolonged process that uses a variety of tools from diverse fields. To accelerate the process, a number of biotechnologies, including genomics, proteomics and a number of cellular and organismic methodologies, have been developed. Proteomics development faces interdisciplinary challenges, including both the traditional (biology and chemistry) and the emerging (high-throughput automation and bioinformatics). Emergent technologies include two-dimensional gel electrophoresis, mass spectrometry, protein arrays, isotope-encoding, two-hybrid systems, information technology and activity-based assays. These technologies, as part of the arsenal of proteomics techniques, are advancing the utility of proteomics in the drug-discovery process.

Animals↗
PubMed2006

Proteomic analysis of cartilage- and bone-associated samples.

The skeleton of the human body is built of cartilage and bone, which are tissues that contain extensive amounts of extracellular matrix (ECM). In bone, inorganic mineral hydroxyapatite forms 50-70% of the whole weight of the tissue. Although the organic matrix of bone consists of numerous proteins, 90% of it is composed of type I collagen. In cartilage, ECM forms a major fraction of the tissue, type II collagen and aggrecans being the most abundant macromolecules. It is obvious that the high content of ECM components causes analytical problems in the proteomic analysis of cartilage and bone, analogous to those in the analysis of low-abundance proteins present in serum. The massive contents of carbohydrates present in cartilage proteoglycans, and hydroxyapatite in bone, further complicate the situation. However, the development of proteomic tools makes them more and more tempting also for research of musculoskeletal tissues. Application of proteomic techniques to the research of chondrocytes, osteoblasts, osteocytes, and osteoclasts in cell cultures can immediately benefit from the present knowledge. Here we make an overview to previous proteomic research of cartilage- and bone-associated samples and evaluate the future prospects of applying proteomic techniques to investigate key events, such as cellular signal transduction, in cartilage- and bone-derived cells.

Amino Acid Sequence↗
PubMed2006

Study of posttranslational modifications in lenticular alphaA-Crystallin of mice using proteomic analysis techniques.

In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.

Aging↗
PubMed2007

Material surfaces affect the protein expression patterns of human macrophages: A proteomics approach.

Monocyte-derived macrophages (MDM) are key inflammatory cells and are central to the foreign body response to implant materials. MDM have been shown to exhibit changes in actin cytoskeleton, multinucleation, cell size, and function in response to small alterations in polycarbonate-urethane (PCNU) surface chemistry. Although PCNU chemistry has an influence on de novo protein synthesis, no assessments of the protein expression profiles of MDM have yet been reported. The rapid emerging field of expression proteomics facilitates the study of changes in cellular protein profiles in response to their microenvironment. The current study applied proteomic techniques, 2-dimensional electrophoresis (2-DE) combined with MALDI-ToF (matrix assisted laser desorption ionization-time of flight) mass spectrometry, to determine differences in MDM protein expression influenced by PCNU. Results indicated that MDM responded to material chemistry by modulation of structural proteins (i.e. actin, vimentin, and tubulin). Additionally, intracellular protein modulation which requires proteins responsible for trafficking (i.e. chaperone proteins) and protein structure modification (i.e. bond rearrangement and protein folding) were also altered. This study demonstrated for the first time that a proteomics approach was able to detect protein expression profile changes in MDM cultured on different material surfaces, forming the basis for utilizing further quantitative proteomics techniques that could assist in elucidation of the mechanisms involved in MDM-material interaction.

Biocompatible Materials↗
PubMed2002

Past, present and future strategies to study the genetics of body weight regulation.

Genetic advances have made remarkable progress towards our understanding of body weight regulation. Much of our current knowledge has come from the cloning and characterisation of the genes responsible for obesity syndromes in the mouse, and the identification of homologous mutations causing rare forms of obesity in humans. Gene targeting experiments in mice have been instrumental in confirming the importance of many genes in the aetiology of obesity, and the existence of a fundamental physiological pathway that controls energy balance is becoming clear. The genetic determinants that underlie common forms of human obesity are largely polygenic, with most genes producing small effects. Thus, elucidating the many genetic determinants of obesity is a current challenge for modern geneticists. Despite the inherent difficulties, progress has been made through linkage/association studies and a genetic map of quantitative trait loci for human obesity is beginning to emerge. Obesity research is now very much in a transition period. Not so long ago, access to high throughput screening, as well as microarray and proteomic techniques, was prohibitively expensive and available only to the few. In recent years, these technologies have become more accessible to the larger scientific community and, in this paper, we will discuss how such technological advances are likely to drive the next wave of progress in obesity research. For example, large-scale mutagenesis screens in rodents coupled with high throughput screening are likely to emerge as important technologies for identifying genes previously unexpected to be involved in body weight regulation. Furthermore, applications of microarray and proteomic techniques will further refine our understanding of currently known peptides as well as identify novel pathways and molecules which are involved in energy homeostasis.

Agouti Signaling Protein↗
PubMed2003

Prefractionation techniques in proteome analysis.

The present review deals with a number of prefractionation protocols in preparation for two-dimensional map analysis, both in the fields of chromatography and in the field of electrophoresis. In the first case, Fountoulaki's groups has reported just about any chromatographic procedure useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins. As a result of the various enrichment steps, several hundred new species, previously undetected in unfractionated samples, could be revealed for the first time. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps. The devices here reviewed include multichamber apparatus, such as the multicompartment electrolyzer with Immobiline membranes, Off-Gel electrophoresis in a multicup device and the Rotofor, an instrument also based on a multichamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other instruments of interest are the Octopus, a continuous-flow device for isoelectric focusing in a upward flowing liquid curtain, and the Gradiflow, where different pI cuts are obtained by a multistep passage through two compartments buffered at different pH values. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".

Chemical Fractionation↗
PubMed2004

Vascular proteomics: linking proteomic and metabolomic changes.

Cardiovascular diseases constitute the largest of death in the Western world. Various stressors, including elevated blood pressure, smoking, diabetes, and hypercholesterolemia directly or indirectly damage the vessel wall, eventually inducing arterial stiffness (arteriosclerosis) and lipid accumulation (atherosclerosis). However, the molecular mechanisms of atheroma formation are not yet fully clarified. While many investigators have used proteomic techniques to study cardiac diseases, vascular proteomics is still in its infancy. The present review highlights studies, in which proteomics has been successfully applied to study protein alterations in the vasculature. Furthermore, we will summarize our recent progress in combining proteomic and metabolomic techniques to reveal protein and metabolite alterations in the cardiovascular system: two-dimensional (2-D) gel electrophoresis proved to be highly complementary to nuclear magnetic resonance (NMR) spectroscopy, in that post-translational modifications of the most abundant enzymes were displayed on 2-D gels while NMR spectroscopy revealed changes in the corresponding metabolites. Importantly, the simultaneous assessment of protein and metabolite changes translated purely descriptive proteomic and metabolomic profiles into a functional context and provided important insights into pathophysiological mechanisms that would not have been obtained by other techniques.

Animals↗
PubMed2006

Proteomic analysis of nitrated and 4-hydroxy-2-nonenal-modified serum proteins during aging.

Using proteomic techniques, we investigated peroxynitrite (ONOO-) and 4-hydroxy-2-nonenal (HNE) modified serum proteins from young and old Fischer 344 rats. Two-dimensional gel electrophoresis/western blot analysis of nitrotyrosine and HNE-histidine revealed that serum proteins were differentially modified by ONOO- and HNE. Among them, 16 of the modified proteins, identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), are involved in blood coagulation, lipid transport, blood pressure regulation, and protease inhibition. Furthermore, nitration and HNE adduction were found to increase with age, lending support to the oxidative stress hypothesis of aging. Our data showed that proteomic techniques can be valuable tools in the study of protein profiling modifications during aging.

Aging↗
PubMed2005

Prefractionation techniques in proteome analysis: the mining tools of the third millennium.

The present review deals with prefractionation protocols used in proteomic investigation in preparation for mass spectrometry (MS) or two-dimensional electrophoresis (2-DE) map analysis. Briefly, reported methods focus on cell organelle differential centrifugation and on chromatographic approaches, to continue in extenso with a panoply of electrophoretic methods. In the case of chromatography, procedures useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins, revealed several hundreds of new species, previously undetected in unfractionated samples. Novel chromatographic prefractionation methods are also discussed such as a multistaged fractionation column, consisting in a set of immobilized chemistries, serially connected in a stack format (an assembly of seven blocks), each capable of harvesting a given protein population. Such a method significantly simplifies the complexity of treated samples while concentrating species, all resulting in a larger number of visible proteins by MS or 2-DE. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps (although some approaches based on gels and granulated media are also discussed). Devices associated with electrophoretic separation are multichamber apparatus, such as the multicompartment electrolyzers equipped with either isoelectric membranes or with isoelectric beads. Multicup device electrophoresis and several others, exploiting the conventional technique of carrier ampholyte focusing, are reviewed. This review also reports approaches for sample treatments in order to detect low-abundance species. Among others, a special emphasis is made on the reduction of concentration difference between proteins constituting a sample. This latter consists in a library of combinatorial ligands coupled to small beads. Such a library comprises hexameric ligands composed of 20 amino acids, resulting in millions of different structures. When these beads are impregnated with complex proteomes (e.g., human sera) of widely differing protein compositions, they are able to significantly reduce the concentration differences, thus greatly enhancing the possibility to evidence low-abundance species. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".

Cell Fractionation↗
PubMed2006

Application of surface-enhanced laser desorption/ionization time-of-flight-based serum proteomic array technique for the early diagnosis of prostate cancer.

AIM: To identify the serum biomarkers of prostate cancer (PCa) by protein chip and bioinformatics. METHODS: Serum samples from 83 PCa patients and 95 healthy men were taken from a mass screening in Changchun, China. Protein profiling was carried out using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The data of spectra were analyzed using two bioinformatics tools. RESULTS: Eighteen serum differential proteins were identified in the PCa group compared with the control group (P < 0.01). There were four proteins at the higher serum level and 14 proteins at the lower serum level in the PCa group. A decision tree classification algorithm that used an eight-protein mass pattern was developed to correctly classify the samples. A sensitivity of 92.0% and a specificity of 96.7% for the study group were obtained by comparing the PCa and control groups. CONCLUSION: We identified new serum biomarkers of PCa. SELDI-TOF MS coupled with a decision tree classification algorithm will provide a highly accurate and innovative approach for the early diagnosis of PCa.

Aged↗
PubMed2006

Pilot study of the Human Proteome Organisation Brain Proteome Project: applying different 2-DE techniques to monitor proteomic changes during murine brain development.

The Human Proteome Organisation Brain Proteome Project aims at coordinating neuroproteomic activities with respect to analysis of development, aging, and evolution in human and mice and at analysing normal aging processes as well as neurodegenerative diseases. Our group participated in the mouse pilot study of this project using two different 2-DE systems, to find out the optimal conditions for comprehensive gel-based differential proteome analysis. Besides the assessment of the best methodical conditions the question of "How many biological replicate analyses have to be performed to get reliable statistically validated results?" was addressed. In total 420 differences were detected in all analyses. Both 2-DE methods were found to be suitable for comprehensive differential proteome analysis. Nevertheless, each of the methods showed substantial advantages and disadvantages resulting in the fact that modification of both systems is essential. From our results we can draw the conclusions that for the future optimal quantitative differential gel-based brain proteome analyses the sample preparation has to be slightly changed, the resolution of the first as well as the second dimension has to be advanced, the number of experiments has to be increased and that the 2D-DIGE system should be applied.

Aging↗