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PubMed2001

Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins.

The number of unique protein species in proteomes from a single mammalian cell type is not well defined but is likely to be at least 10000-20000. Since standard-size two-dimensional gels typically resolve only about 1500 to 3000 spots, they merely analyze a small portion of these proteomes. In addition, all insoluble proteins and typically proteins > 100 kDa are seldom resolved on two-dimensional (2-D) gels. The current study demonstrates the feasibility of an overall strategy for more comprehensive quantitative comparisons of complex proteomes derived from physiological fluids or mammalian cell extracts. A key feature of this approach is to prefractionate samples into a few well-resolved fractions based on the proteins' isoelectric points (pIs) using microscale solution isoelectric focusing. These fractions are then separated on narrow pH range two-dimensional gels approximately +/- 0.1 pH unit wider than the prefractionated pool. When this prefractionation approach is applied to complex mammalian proteomes, it improves resolution and spot recovery at high protein loads compared with use of parallel narrow pH range gels without prefractionation. The minimal cross-contamination between fractions allows quantitative comparisons in contrast to most alternative prefractionation methods. In addition, complementary data can be obtained by parallel analysis of the solubilized fraction on high-resolution large-pore-gradient one-dimensional gels followed by mass spectrometric identification to analyze proteins between 100 and approximately 500 kDa. Similarly, insoluble proteins can be analyzed using large-pore gels for large proteins and 10-12% one-dimensional sodium dodecyl sulfate (SDS) gels for smaller proteins. Together, these strategies should permit more reliable quantitative comparisons of complex mammalian proteomes where detection of at least 10000 protein spots is needed in order to analyze the majority of the unique protein species.

Animals↗
PubMed2002

Expression variance, biochemical and immunological properties of Toxoplasma gondii dense granule protein GRA7.

During intracellular stay, Toxoplasma gondii secretes dense granule proteins (GRA) which remodel the parasitophorous vacuole and are considered functional in parasite-host interrelation. Comparative analysis of parasites from mouse-virulent strain BK and an in vitro attenuated variant revealed that the level of GRA7 expression correlates with T. gondii virulence: proteome analysis and quantitation by immunoblot demonstrated a massive decrease in GRA7 steady-state synthesis parallel to the loss of virulence. Properties of GRA7 that are pertinent to its membrane targeting and to GRA7-directed immune resistance were studied in detail. GRA7 is exclusively membrane-associated in both parasites and infected host cells as demonstrated by subcellular fractionations. Triton X-114 partitioning of isolated parasites substantiated that GRA7 is an integral membrane protein, the hydrophobic stretch from amino acid 181 to 202 providing a possible membrane anchor. A fraction enriched for membranous material from infected host cells contained additional forms of GRA7 with reduced mobility in gel electrophoresis, indicating that the protein is modified after exocytosis from the parasite. By flow cytometric analysis, GRA7 was detected on the surface of intact host cells. An intracellular origin of surface-associated GRA7 seems likely since GRA7 released from extracellular parasites failed to label the host cell surface. Consistent with a role at a parasite-host interface, GRA7 proved to be a target antigen of the intracerebral immune response as evidenced by the presence of GRA7-specific antibodies in mouse cerebrospinal fluid during chronic infection.

Animals↗
PubMed2003

Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes.

Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic beta-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.

Adenosine Triphosphate↗
PubMed2006

A general precursor ion-like scanning mode on quadrupole-TOF instruments compatible with chromatographic separation.

MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode--implemented on a quadrupole-TOF instrument--that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain.

Acetylation↗
PubMed2025

MEG3 Promoter Methylation and F11 Receptor (F11R) Overexpression Define a High-Risk Subtype of Diabetic Pancreatic Cancer.

Long-standing diabetes mellitus (long-DM) (≧3 years) is associated with worse clinical outcomes in patients with pancreatic ductal adenocarcinoma (PDAC). Emerging evidence suggests that epigenetic alterations may contribute to this association; however, the underlying mechanisms remain largely unclear. This study aimed to elucidate the role of the tumor-suppressive long noncoding RNA maternally expressed gene 3 (MEG3) and related molecules in the development of PDAC with long-DM. A total of 117 patients who underwent surgical resection for PDAC at Hirosaki University Hospital were retrospectively analyzed. Histopathological assessment followed World Health Organization criteria and the Union for International Cancer Control tumor-node-metastasis classification. Promoter methylation of MEG3 was assessed via methylation-specific PCR using formalin-fixed paraffin-embedded tissue. MEG3 expression levels were assessed by real-time quantitative PCR. Additionally, proteomic profiling was performed using liquid chromatography-tandem mass spectrometry on formalin-fixed paraffin-embedded tissue samples. Among the 117 cases with PDAC, patients with long-DM exhibited significantly poorer tumor differentiation and reduced cancer-specific survival. MEG3 promoter methylation was more prevalent in patients with long-DM. MEG3 methylation was correlated with reduced MEG3 expression, increased venous invasion, higher recurrence rates, and worse prognosis. Proteomic analysis and protein structure prediction tool revealed F11 receptor (F11R) as a potential downstream effector of MEG3. F11R protein expression levels were evaluated using semiquantitative immunohistochemistry. Higher F11R expression was observed in patients with long-DM, correlating with poor histologic differentiation and unfavorable outcomes. Patients with PDAC showing simultaneous MEG3 methylation and F11R high expression were more likely to have long-DM, with additive effects of these changes and tumor recurrence. Our results demonstrated that MEG3 and its potential downstream regulator, F11R, could be involved in PDAC progression, particularly in patients with long-DM. The findings underscore the clinical significance of epigenetic regulation in DM-related PDAC, suggesting novel targets, such as MEG3 and F11R, for potential therapeutic intervention.

Humans↗
PubMed2026

Uncovering the signaling networks of disseminated glioblastoma cells in vivo with INSIGHT.

Dysregulation of intracellular signaling networks underpins cancer. Yet, resolving signaling networks within distinct or rare cell types in cancer in vivo has been unattainable. Here we develop INSIGHT by integrating cell sorting with mass spectrometry to enable quantitative phosphoproteomics and proteomics of discrete cell types from fixed tissues. Using INSIGHT, we map the signaling network within disseminating glioblastoma cells from patient-derived xenografts implanted in mice. Disseminating tumor cells undergo a proteome-wide shift from proliferative to mesenchymal, neural progenitor-like cell states. In parallel, signaling network and global kinase activity are rewired, transitioning from cell cycle-associated circuitries to those governing synaptic function, neuronal migration, and ion channel activity. Changes begin at the tumor margin and persist in distant brain parenchyma. Hornerin and phosphorylation of Ca²⁺-permeable GluA2 at Y876 were identified as mediators of glioblastoma progression. INSIGHT enables systems-level dissection of cell-type-specific signaling circuitries in vivo across wide range of biological systems.

Glioblastoma↗
PubMed2005

Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model.

With advancements in the analytical technologies and methodologies in proteomics, there is great interest in biomarker discovery in biofluids such as serum and plasma. Current hypotheses suggest that the low molecular weight (LMW) serum proteome possesses an archive of clipped and cleaved protein fragments that may provide insight into disease development. Though these biofluids represent attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. Mice are one of the most extensively used animal models for studying human disease because they represent a highly controllable experimental model system. In this study, the LMW serum proteome was compared between xenografted tumor-bearing mice and control mice by differential labeling utilizing trypsin-mediated incorporation of the stable isotope of oxygen, 18O. The digestates were combined, fractionated by strong cation exchange chromatography, and analyzed by nanoflow reversed-phase liquid chromatography coupled online with tandem mass spectrometry, resulting in the identification of 6003 proteins identified by at least a single, fully tryptic peptide. Almost 1650 proteins were identified and quantitated by two or more fully tryptic peptides. The methodology adopted in this work provides the means for future quantitative measurements in comparative animal models of disease and in human disease cohorts.

Animals↗
PubMed2002

Combinatorial use of mRNA and two-dimensional electrophoresis expression data to choose relevant features for mass spectrometric identification.

It is only recently that quantitative studies of differential proteome analysis (DPA) have become possible. In this paper the issues involved in quantitative DPA are discussed and novel tools to select features for identification by mass spectrometry (MS) are described. The problem of comparing two sets of gels on a global level is explored as well as how to find specific protein features that differentiate two sets of two-dimensional electrophoresis gels. The concept of a 'virtual' gel, derived from gene expression data, is introduced. The virtual gel enables the co-analysis of data from gene and protein expression. We discuss the value of such an approach, and consider what new information can be gained by using gene and protein expression together. These tools are illustrated by analysis of data from tandem gene and protein expression experiments. Features that are highlighted by the above methods are putative candidates for MS identification. Tools are described that integrate the process of feature selection, cutting, and MS analysis.

Breast↗
PubMedDate not supplied

Quantitative profiling of the detergent-resistant membrane proteome of iota-b toxin induced vero cells.

Enzyme-mediated 18O/16O differential labeling of proteome samples often suffers from incomplete exchange of the carboxy-terminus oxygen atoms, resulting in ambiguity in the measurable abundance differences. In this study, an 18O/16O labeling strategy was optimized for and applied to the solution-based comparative analysis of the detergent-resistant membrane proteome (DRMP) of untreated and Iota-b (Ib)-induced Vero cells. Solubilization and tryptic digestion of the DRMP was conducted in a buffer containing 60% methanol. Unfortunately, the activity of trypsin is attenuated at this methanol concentration hampering the ability to obtain complete oxygen atom turnover. Therefore, the incorporation of the 18O atoms was decoupled from the protein digestion step by carrying out the trypsin-mediated heavy atom incorporation in a buffer containing 20% methanol; a concentration at which trypsin activity is enhanced compared to purely aqueous conditions. After isotopic labeling, the samples were combined, fractionated by strong cation exchange and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. In total, over 1400 unique peptides, corresponding to almost 600 proteins, were identified and quantitated, including all known caveolar and lipid raft marker proteins. The quantitative profiling of Ib-induced DRMP from Vero cells revealed several proteins with altered expression levels suggesting their possible role in Ib binding/uptake.

ADP Ribose Transferases↗
PubMed2005

Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.

Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. Development of sensitive and comprehensive analytical methods for determination of protein phosphorylation is therefore a necessity in the pursuit of a detailed molecular view of complex biological processes. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for identification. This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. SILAC encoding of yeast proteomes was achieved by incorporation of [(13)C(6)]arginine and [(13)C(6)]lysine in a double auxotroph yeast strain. Pheromone-treated yeast cells were mixed with SILAC-encoded cells as the control and lysed, and extracted proteins were digested with trypsin. Phosphopeptides were enriched by a combination of strong cation exchange chromatography and IMAC. Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified phosphopeptides, 139 were differentially regulated at least 2-fold in response to mating pheromone. Among these regulated proteins were components belonging to the mitogen-activated protein kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth, and the regulation of the cell cycle.

Amino Acid Sequence↗
PubMed2004

Proteomics: current techniques and potential applications to lung disease.

Proteomics aims to study the whole protein content of a biological sample in one set of experiments. Such an approach has the potential value to acquire an understanding of the complex responses of an organism to a stimulus. The large vascular and air space surface area of the lung expose it to a multitude of stimuli that can trigger a variety of responses by many different cell types. This complexity makes the lung a promising, but also challenging, target for proteomics. Important steps made in the last decade have increased the potential value of the results of proteomics studies for the clinical scientist. Advances in protein separation and staining techniques have improved protein identification to include the least abundant proteins. The evolution in mass spectrometry has led to the identification of a large part of the proteins of interest rather than just describing changes in patterns of protein spots. Protein profiling techniques allow the rapid comparison of complex samples and the direct investigation of tissue specimens. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. These methodologies have made the application of proteomics on the study of specific diseases or biological processes under clinically relevant conditions possible. The quantity of data that is acquired with these new techniques places new challenges on data processing and analysis. This article provides a brief review of the most promising proteomics methods and some of their applications to pulmonary research.

Animals↗
PubMed2005

Proteomic analysis of different mutant genotypes of Arabidopsis led to the identification of 11 proteins correlating with adventitious root development.

A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis (Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities.

Adaptation, Physiological↗
PubMed2005

Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets.

For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH-precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two-dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH- and TCA-precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.

Adult↗
PubMed2005

Proteomics.

Proteomics is the measurement of one or more protein populations or proteomes, preferably in a quantitative manner. A protein population may be the set of proteins found in an organism, in a tissue or biofluid, in a cell, or in a subcellular compartment. A population also may be the set of proteins with a common characteristic, for example, those that interact with each other in molecular complexes, those involved in the same process such as signal transduction or cell cycle control, or those that share a common posttranslational modification such as phosphorylation or glycosylation. Proteomics experiments that involve mass spectrometry are divided into five categories: (1) protein identification, (2) protein quantitation or differential analysis, (3) protein-protein interactions, (4) post-translational modifications, and (5) structural proteomics. Each of these proteomics categories is reviewed. Examples are given for quantitative experiments involving two-dimensional gel electrophoresis, and for gel-free analysis using isotope-coded affinity tags. The impact of proteomics on biological research and on drug development is discussed. Challenges for further development in proteomics are presented, including sample preparation, sensitivity, dynamic range, and automation.

Animals↗
PubMed2006

Proteomic technology for biomarker profiling in cancer: an update.

The progress in the understanding of cancer progression and early detection has been slow and frustrating due to the complex multifactorial nature and heterogeneity of the cancer syndrome. To date, no effective treatment is available for advanced cancers, which remain a major cause of morbidity and mortality. Clearly, there is urgent need to unravel novel biomarkers for early detection. Most of the functional information of the cancer-associated genes resides in the proteome. The later is an exceptionally complex biological system involving several proteins that function through posttranslational modifications and dynamic intermolecular collisions with partners. These protein complexes can be regulated by signals emanating from cancer cells, their surrounding tissue microenvironment, and/or from the host. Some proteins are secreted and/or cleaved into the extracellular milieu and may represent valuable serum biomarkers for diagnosis purpose. It is estimated that the cancer proteome may include over 1.5 million proteins as a result of posttranslational processing and modifications. Such complexity clearly highlights the need for ultra-high resolution proteomic technology for robust quantitative protein measurements and data acquisition. This review is to update the current research efforts in high-resolution proteomic technology for discovery and monitoring cancer biomarkers.

Biomarkers, Tumor↗
PubMed2006

Toward the application of proteomics to human thyroid tissue.

CONTEXT: In this paper we describe for the first time a systematic approach to proteome analysis of human thyroid tissue. OBJECTIVE AND DESIGN: We report different methods to decrease the complexity of the human thyroid tissue proteome by applying different solubilization strategies and correcting for thyroglobulin protein abundance; to increase the protein resolution by prefractionation and by the use of narrow-range pH gradients; to detect proteins using sensitive and quantitative stains; and to identify soluble and membrane-bound thyroid tissue proteins by mass spectrometry analysis. MAIN OUTCOME/RESULTS: We found that buffers containing high contents of urea and detergents allow the best solubilization of human thyroid tissue proteins; highly variable abundance of thyroglobulin is a major pitfall of human thyroid proteome analysis, which in contrast to centrifugal ultrafiltration, size-exclusion chromatography and microdissection, can be countered best by adapting the protein amount to the thyroglobulin content per sample; prefractionation leads to a significant enrichment of proteins and allows subcellar localization of thyroid proteins; application of narrow-range immobilized pH gradient (IPG) strips allows further improvement of spot detection and separation; and protein detection with the fluorescent stain ruthenium II Tris bathophenanthroline disulfonate (RuBPs) is a highly sensitive and reliable tool for quantitative proteome analysis. Finally, in a pilot study of four patients with benign nodular thyroid disease we found that the described procedures allow a highly reproducible detection and identification of alterations in protein expression between nodular and corresponding normal thyroid tissues. CONCLUSIONS: Application of the described methods provides the basis for a highly sensitive and reproducible proteome analysis of the human thyroid, providing an additional novel tool to elucidate complex proteins changes in human thyroid biology as well as pathophysiology of human thyroid disease.

Cell Fractionation↗
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↗
PubMed2004

Genotypic and phenotypic biomarker profiles for individual risk assessment and cancer detection (lessons from bladder cancer risk assessment in symptomatic patients and workers exposed to benzidine).

There is a need for improved methods for detecting individuals at risk for cancer to target subsets of patients for more intensive individual screening and targeted cancer therapy and chemoprevention. One approach for accomplishing this objective is to detect premalignant molecular fingerprints in an organ at risk for cancer or to define biomarkers reflective of treatment selection and response. Bladder cancer is an excellent model for testing this approach; however, comprehending the strategy for biomarker selection and analysis is more complicated than is generally appreciated. The objective of this article is to provide a succinct overview of our experience with the selection of biomarkers for bladder cancer detection, first in symptomatic patients and then in high-risk cohorts of workers at risk for bladder cancer. Biomarker selection depends on multiple parameters, each of which must be optimized to enhance the utility of a biomarker for clinical application. Many markers that initially show promise fail in the clinical arena for a variety of reasons. Important parameters include when a biomarker is expressed in carcinogenesis (i.e., early vs. late), the sample type, and the method of analysis. These all contribute to the sensitivity, specificity, and ultimate clinical utility of a biomarker. New technologies/ support the notion that all diseases start in the cell, and Seymore West indicated the cell, under appropriate conditions, can function as a microcuvette for biophysical cytochemical analysis. Spectroscopy provides an accurate and sensitive method for quantitative single-cell proteomics. Improved and more stable fluorescence probes will enhance the utility of cellular chemistry, as will a rationale approach for biomarker selection based on the concepts of field cancerization, complemented by improved quantitative analysis of protein markers at the single-cell level. Our laboratory has developed a platform for single-cell proteomic analysis that can be applied to multiple basic science and clinical problems. Single-cell proteomics also facilitates the study of genetic instability and epigenetic signaling (stromal-epithelial interactions) in relation to cancer therapy and diagnosis. Because most cancers arise through multiple signaling pathways and are heterogeneous, the identification of appropriate biomarker profiles provides a number of strategic advantages over a single biomarker. Complex networks of signaling pathways lead to increased cell proliferation, decreased cell adhesion, cellular differentiation, genetic instability, and other functions associated with the malignant phenotype. The purpose of this presentation is to illustrate the fundamental concepts for selection and profile analysis of high-level phenotypic biomarkers developed for bladder cancer risk assessment, screening, and early bladder cancer detection.

Benzidines↗