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At least 1,207 records · Page 67Linked to original sources
PubMed2004

Quantitative profiling of LNCaP prostate cancer cells using isotope-coded affinity tags and mass spectrometry.

Androgens are involved in the pathogenesis of diseases including adenocarcinoma of the prostate. These hormones are important for growth, maintenance, and integrity of structure and function of the prostate. Androgen-deprivation is currently the only effective systemic therapy for prostate cancer but the effects of androgens on the proteome are still poorly described. Here we quantitatively profile changes in the proteome of LNCaP human prostate cancer cells in response to androgen using the newly developed isotope-coded affinity tag (ICAT) labeling and two-dimensional liquid chromatography-tandem mass spectroscopy (2-D LC-MS/MS). ICAT enables the concurrent identification and comparative quantitative analysis of proteins present in various biological samples including human cell and tissue extracts. Quantification and identification of 139 proteins in complex protein mixtures obtained from androgen-stimulated and unstimulated LNCaP cells were achieved. Changes in levels of 77 proteins in response to androgens were detected. Some of these proteins have been previously reported to be regulated by androgens and include spermine synthase, fatty acid synthase and calreticulin precursor. A large number of proteins that have not been previously reported to be expressed in prostate cells were also quantitatively identified. Examples of these include members of the dual specificity protein phosphatase subfamily, "similar" to hypothetical protein DKFZp434B0328.1, "similar" to 14-3-3 protein zeta and "similar" to hypothetical protein 458, components of the actin cytoskeleton and a range of unknown/uncharacterized proteins. This catalogue of proteins provides an overview of the proteome of prostate cancer cells and the global changes that occur in response to androgens.

Androgens↗
PubMed2024

Chemical proteomics to study metabolism, a reductionist approach applied at the systems level.

Cellular metabolism encompasses a complex array of interconnected biochemical pathways that are required for cellular homeostasis. When dysregulated, metabolism underlies multiple human pathologies. At the heart of metabolic networks are enzymes that have been historically studied through a reductionist lens, and more recently, using high throughput approaches including genomics and proteomics. Merging these two divergent viewpoints are chemical proteomic technologies, including activity-based protein profiling, which combines chemical probes specific to distinct enzyme families or amino acid residues with proteomic analysis. This enables the study of metabolism at the network level with the precision of powerful biochemical approaches. Herein, we provide a primer on how chemical proteomic technologies custom-built for studying metabolism have unearthed fundamental principles in metabolic control. In parallel, these technologies have leap-frogged drug discovery through identification of novel targets and drug specificity. Collectively, chemical proteomics technologies appear to do the impossible: uniting systematic analysis with a reductionist approach.

Humans↗
PubMed2005

The nuclear membrane proteome: extending the envelope.

The marriage of proteomics with cell biology has produced extensive inventories of the proteins that inhabit several subcellular organelles. Recent proteomic analysis has identified many new putative transmembrane proteins in the nuclear envelope, and transcriptome profiling suggests that the nuclear-membrane proteome exhibits some significant variations among different tissues. Cell-type-specific differences in the composition of protein sub-complexes of the nuclear envelope, particularly those containing the disease-associated protein lamin A, could yield distinctive functions and, thus, explain the tissue specificity of a diverse group of nuclear-envelope-linked disorders in humans. Considered together, these recent results suggest an unexpected functional complexity at the nuclear envelope.

Animals↗
PubMed2004

MitoP2, an integrated database on mitochondrial proteins in yeast and man.

The aim of the MitoP2 database (http://ihg.gsf.de/mitop2) is to provide a comprehensive list of mitochondrial proteins of yeast and man. Based on the current literature we created an annotated reference set of yeast and human proteins. In addition, data sets relevant to the study of the mitochondrial proteome are integrated and accessible via search tools and links. They include computational predictions of signalling sequences, and summarize results from proteome mapping, mutant screening, expression profiling, protein-protein interaction and cellular sublocalization studies. For each individual approach, specificity and sensitivity for allocating mitochondrial proteins was calculated. By providing the evidence for mitochondrial candidate proteins the MitoP2 database lends itself to the genetic characterization of human mitochondriopathies.

Computational Biology↗
PubMed2004

Proteomic analysis finds different myocardial protective mechanisms for median nerve stimulation by electroacupuncture and by local somatothermal stimulation.

Ischemia-reperfusion (I/R) injury of the heart can be attenuated by application of median nerve stimulation (MNS) through either electroacupuncture (EA) or local somatothermal stimulation (LSTS). This study is aimed to investigate the differences of myocardial protein expression between MNS by EA and by LSTS. Twenty-four male Sprague-Dawley rats were randomly allocated to 8 groups. In MNS by EA groups, animals were EA-stimulated for 30 min and the hearts were taken for proteomics analysis at an interval of 0 min (group 1), 30 min (group 2), 60 min (group 3) and control group (group 4); whereas in MNS by LSTS groups, following 1 dose (group 5), 2 doses (group 6), 3 doses (group 7) and control group (group 8) of LSTS, the hearts were taken for proteomics analysis. Myocardial protein expression profiles were analyzed and validated by Western blot analysis. Finally, the biological responses were also evaluated. The results showed that either MNS by EA followed by a 30-min rest period (group 2) and by 3 doses of LSTS (group 7) had cardioprotective effect against I/R injury. However, the myocardial protein expression profiles are quite different between the above-mentioned groups. We conclude that MNS by either EA or LSTS attenuates I/R injury in rat hearts through different protective mechanisms and that EA and LSTS might provide an alternative treatment strategy for ischemic heart disease.

Animals↗
PubMed2005

Unraveling the pathogenesis of type 1 diabetes with proteomics: present and future directions.

Type 1 diabetes (T1D) is the result of selective destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. T1D is due to a complex interplay between the beta-cell, the immune system, and the environment in genetically susceptible individuals. The initiating mechanism(s) behind the development of T1D are largely unknown, and no genes or proteins are specific for most T1D cases. Different pro-apoptotic cytokines, IL-1 beta in particular, are present in the islets during beta-cell destruction and are able to modulate beta-cell function and induce beta-cell death. In beta-cells exposed to IL-1 beta, a race between destructive and protective events are initiated and in susceptible individuals the deleterious events prevail. Proteins are involved in most cellular processes, and it is thus expected that their cumulative expression profile reflects the specific activity of cells. Proteomics may be useful in describing the protein expression profile and thus the diabetic phenotype. Relatively few studies using proteomics technologies to investigate the T1D pathogenesis have been published to date despite the defined target organ, the beta-cell. Proteomics has been applied in studies of differentiating beta-cells, cytokine exposed islets, dietary manipulated islets, and in transplanted islets. Although that the studies have revealed a complex and detailed picture of the protein expression profiles many functional implications remain to be answered. In conclusion, a rather detailed picture of protein expression in beta-cell lines, islets, and transplanted islets both in vitro and in vivo have been described. The data indicate that the beta-cell is an active participant in its own destruction during diabetes development. No single protein alone seems to be responsible for the development of diabetes. Rather the cumulative pattern of changes seems to be what favors a transition from dynamic stability in the unperturbed beta-cell to dynamic instability and eventually to beta-cell destruction.

Animals↗
PubMed2003

Mass spectrometry-based clinical proteomics.

In recent years, mass spectrometry (MS) has been recognized as a 'Gold Standard' tool for the identification and analysis of individual proteins in expression proteomics studies. Moreover, MS has proven useful for the analysis of nucleic acids for single nucleotide polymorphism (SNP) genotyping purposes. With the increased usage of MS as a standard tool for life science applications and the advancement of MS instrumentation, sample preparation and bioinformatics, MS technology has entered novel screening and discovery application areas that are beyond the traditional protein identification and characterization applications. The areas of clinical diagnostics and predictive medicine are just two prime examples of these fields. Predictive markers or biomarkers for early diagnosis of diseases are of growing importance for the human healthcare community. The goal of using MS in clinical proteomics is to generate protein profiles (mass to charge [m/z] ratio versus signal intensity) from readily available body fluids like serum, saliva and urine to detect changes in protein levels that reflect changes in the disease states. Whereas the results originating from individual protein markers may be intriguing, data resulting from the analysis of complex, multiple biomarker patterns may be unequivocal. These biomarker patterns are hidden in complex mass spectra and are not always obvious to the human eye. Sophisticated bioinformatics algorithms have to be applied to determine these unique biomarker patterns. Here, we review the latest developments concerning the use of MS for the discovery of biomarker patterns and for the identification of individual biomarkers in the field of clinical proteomics applications.

Biomarkers↗
PubMed2025

A comprehensive analysis of supermere, exomere, and extracellular vesicle isolation and cargo in colorectal cancer.

Biofluids contain a heterogeneous mixture of extracellular vesicles and non-vesicular nanoparticles (including exomeres and supermeres) that transport a diverse array of proteins, RNA, and lipids. Our previous efforts to characterize the contents of these carriers in colorectal cancer relied on 2D culture systems requiring large-scale setups and time-consuming ultracentrifugation-based isolation. To streamline this process, we have combined 3D hollow-fiber bioreactor production and fast-protein liquid chromatography-based size-exclusion chromatography. Here, we compare the impact of culture methods and purification strategies on small extracellular vesicle, exomere, and supermere cargo. Proteomic analyses show consistently distinct profiles for extracellular vesicles, exomeres, and supermeres regardless of culture conditions or isolation method. In contrast, these two variables influence small RNAs, their base modifications, and lipidomic profiles. We present an online tool to query these and future secretome datasets (https://superomics.shinyapps.io/browse).

Humans↗
PubMed2002

Quantitative protein profiling using two-dimensional gel electrophoresis, isotope-coded affinity tag labeling, and mass spectrometry.

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.

Affinity Labels↗
PubMed2005

Throwing new light on lung cancer pathogenesis: updates on three recent topics.

Lung cancers have become the leading cause of cancer deaths in Japan, claiming more than 55 000 lives annually. Unfortunately, substantial improvement in terms of cure rates has not been achieved over the last two decades, although during the same period of time in-depth basic knowledge of the molecular mechanisms, which underlies carcinogenesis and progression of this deadly group of neoplasms, has accumulated at an amazing pace. It has consequently become evident that they have many shared but also distinct features, when comparisons are made not only with other common epithelial cancers of adults, such as colon cancer, but also within the various histologic types of lung cancers themselves. This review article provides an up-date on cutting-edge research into the following three different topics, from which important new insights have been obtained. The first concerns genetic instability, especially chromosome instability, and checkpoint failure in lung cancers. Second, we deal with EGFR mutations, which shows revealing specificities in various aspects. Finally, advances in the expression profiling analysis of both transcriptomes and proteomes of lung cancers are summarized.

Adenocarcinoma↗
PubMed2026

Plasma proteomics and incident coronary heart disease.

BACKGROUND: Systematic profiling of plasma proteins in population studies offers a complementary approach to discovery of novel risk factors and may provide new insights into the causes of coronary heart disease. METHODS: To explore relationships between the circulating proteome and coronary heart disease (CHD), we evaluated associations of 4780 plasma proteins with incident CHD in the Cardiovascular Health Study (CHS, N=2856, 575 CHD events) and replicated significant associations in the Atherosclerosis Risk in Communities Study (ARIC, N = 10456; 1375 events). RESULTS: We find that 11 proteins significantly associate with incident CHD after adjusting for risk factors; and eight significantly replicated in ARIC. Several proteins correlate with carotid intimal medial thickness and CHD associations are attenuated in participants without subclinical atherosclerosis. Macrophage metalloelastase (MMP12) is the strongest observed association (Hazard Ratio, 1.31; 95% Confidence Interval, 1.19-1.44). Mendelian randomization (MR) identifies a causal relationship between higher MMP12 and lower CHD (Odds Ratio, OR 0.94) and ischemic stroke (OR 0.90) risk, while reverse MR found that genetic propensity to CHD increased MMP12. Taken together, multivariable MR confirms a direct protective effect of higher plasma MMP12 on CHD risk and a genetic effect of atherosclerosis and CHD on elevating MMP12. CONCLUSIONS: Proteomic analyses reveal associations with incident CHD and genomic evidence suggests that therapeutic MMP12 inhibition may confer adverse cardiovascular effects.

Journal Article↗
PubMed2006

Modern genome-wide genetic approaches to reveal intrinsic properties of stem cells.

PURPOSE OF REVIEW: The clinical use of hematopoietic stem cells, which produce all mature blood cell lineages in the circulation, is continuously increasing. Identification of genes and gene networks specifying either stemness or commitment will not only be of major relevance for a fundamental understanding of developmental biology, but also for the emerging fields of tissue engineering and regenerative medicine. Our appreciation of the transcriptional machinery that distinguishes stem cells from their nonstem cell progeny is, however, rudimentary. State-of-the art genome-wide tools are now becoming available to elucidate intrinsic properties of stem cells. Here, we review recent progress that has been made in this field. RECENT FINDINGS: Approaches to study stem cell-specific genes and gene networks include genetical genomics, mRNA and microRNA expression profiling of carefully selected cells, proteomics, chromatin studies using 'CHIP-on-chip' tools, genome-wide binding site analyses for transcription factors and chromatin-remodeling proteins, and tools to study the three-dimensional organization of gene loci. It is promising to see that the combined application of these tools has resulted in the identification of multiple novel genes that regulate stem cell self-renewal. SUMMARY: Exploitation of the available technology and integrating the data by translation into a dynamic model of networks, operating in all four dimensions, will be essential to fully comprehend the elusive concept of 'stemness'. It is time to harvest.

Animals↗
PubMed2001

Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding.

Reversible tyrosine phosphorylation plays a crucial role in signal transduction, regulating many biological functions including proliferation, differentiation, and motility. The comprehensive characterization of the tyrosine phosphorylation state of a cell is of great interest for understanding the mechanisms that underlie signaling; however, current methods for analyzing tyrosine-phosphorylated proteins in crude protein extracts provide limited information, or are laborious and require relatively large amounts of protein. We have developed a simple, rapid, and flexible competitive binding assay based on the far-Western blot technique, in which a battery of Src homology 2 domain probes is used to detect patterns of specific tyrosine-phosphorylated sites. We demonstrate that distinct profiles of tyrosine phosphorylation can be detected with high sensitivity and specificity and low background. This proteomic approach can be used to rapidly profile the global tyrosine phosphorylation state of any cell of interest and has obvious applications as a molecular diagnostic tool, for example in the classification of tumors. The general strategy we describe here is not limited to Src homology 2 domains and could be used to profile the binding sites for any class of protein interaction domain.

Animals↗
PubMed2006

In-depth analysis of the membrane and cytosolic proteome of red blood cells.

In addition to transporting oxygen and carbon dioxide to and from the tissues, a range of other functions are attributed to red blood cells (RBCs) of vertebrates. Diseases compromising RBC performance in any of these functions warrant in-depth study. Furthermore, the human RBC is a vital host cell for the malaria parasite. Much has been learned from classical biochemical approaches about RBC composition and membrane organization. Here, we use mass spectrometry (MS)-based proteomics to characterize the normal RBC protein profile. The aim of this study was to obtain the most complete and informative human RBC proteome possible by combining high-accuracy, high-sensitivity protein identification technology (quadrupole time of flight and Fourier transform MS) with selected biochemical procedures for sample preparation. A total of 340 membrane proteins and 252 soluble proteins were identified, validated, and categorized in terms of subcellular localization, protein family, and function. Splice isoforms of proteins were identified, and polypeptides that migrated with anomalously high or low apparent molecular weights could be grouped into either ubiquitinylated, partially degraded, or ester-linked complexes. Our data reveal unexpected complexity of the RBC proteome, provide a wealth of data on its composition, shed light on several open issues in RBC biology, and form a departure point for comprehensive understanding of RBC functions.

Cytosol↗
PubMed2003

A reference map of a human pituitary adenoma proteome.

In order to compare the proteomes from different cell types of pituitary adenomas for our long-term goal to clarify the molecular mechanisms that participate in the formation of pituitary adenoma, and to detect any tumor-related marker for an "early-stage" diagnosis, the two-dimensional gel electrophoresis (2-DE) reference map of a pituitary adenoma tissue proteome is described here. A vertical, two-dimensional (2-D) polyacrylamide gel electrophoresis system and PDQuest image analysis software have been used to provide a high level of between-gel reproducibility and to accurately array each protein expressed in a pituitary adenoma tissue. Mass spectrometry (matrix-assisted laser desorption/ionization-time of flight MALDI-TOF and liquid chromatography-electrospray ionization-quadrupole-ion trap LC-ESI-Q-IT) and protein databases were used to characterize each protein in the 2-D gel. The results demonstrate that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among four 2-D gels was 1.95 +/- 0.45 mm in the isoelectric focusing direction, and 1.70 +/- 0.53 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. A total of ca. 1000 protein spots were separated by 2-DE, and 135 protein spots that represent 111 proteins were characterized with mass spectrometry (96 spots for MALDI-TOF, 39 spots for LC-ESI-Q-IT). The characterized proteins include pituitary hormones, cellular signals, enzymes, cellular-defense proteins, cell-structure proteins, transport proteins, etc. Those proteins were located in the cytoplasmic, cellular membrane, mitochondrial, endoplasmic reticulum, nuclear, ribonucleosome, extracellular fractions, or were secreted in plasma, etc. Those identified proteins contribute to a functional profile of the pituitary adenoma proteome. These data will be used to expand the proteome database of the human pituitary, which can be accessed in the website http://www.utmem.edu /proteomics.

Adenoma↗
PubMed2006

Statistically integrated metabonomic-proteomic studies on a human prostate cancer xenograft model in mice.

A novel statistically integrated proteometabonomic method has been developed and applied to a human tumor xenograft mouse model of prostate cancer. Parallel 2D-DIGE proteomic and 1H NMR metabolic profile data were collected on blood plasma from mice implanted with a prostate cancer (PC-3) xenograft and from matched control animals. To interpret the xenograft-induced differences in plasma profiles, multivariate statistical algorithms including orthogonal projection to latent structure (OPLS) were applied to generate models characterizing the disease profile. Two approaches to integrating metabonomic data matrices are presented based on OPLS algorithms to provide a framework for generating models relating to the specific and common sources of variation in the metabolite concentrations and protein abundances that can be directly related to the disease model. Multiple correlations between metabolites and proteins were found, including associations between serotransferrin precursor and both tyrosine and 3-D-hydroxybutyrate. Additionally, a correlation between decreased concentration of tyrosine and increased presence of gelsolin was also observed. This approach can provide enhanced recovery of combination candidate biomarkers across multi-omic platforms, thus, enhancing understanding of in vivo model systems studied by multiple omic technologies.

Animals↗
PubMed2004

Genome-based proteomics.

Protein-protein interactions play crucial roles in various biological pathways and functions. Therefore, the characterization of protein levels and also the network of interactions within an organism would contribute considerably to the understanding of life. The availability of the human genome sequence has created a range of new possibilities for biomedical research. A crucial challenge is to utilize the genetic information for better understanding of protein distribution and function in normal as well as in pathological biological processes. In this review, we have focused on different platforms used for systematic genome-based proteome analyses. These technologies are in many ways complementary and should be seen as various ways to elucidate different functions of the proteome.

Chromatography, Liquid↗