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PubMed2005

Mass spectrometry in diagnostic oncoproteomics.

Diagnostic oncoproteomics is the application of proteomic techniques for the diagnosis of malignancies. A new mass spectrometric technology involves surface enhanced laser desorption ionization combined with time-of flight mass analysis (SELDI-TOF-MS), using special protein chips. After the description of the relevant principles of the technique, including approaches to proteomic pattern diagnostics, applications are reviewed for the diagnosis of ovarian, breast, prostate, bladder, pancreatic, and head and neck cancers, and also several other malignancies. Finally, problems and prospects of the approach are discussed.

Female↗

PubMed2003

Kinome analysis of host response to mycobacterial infection: a novel technique in proteomics.

An array of mammalian phospho-specific antibodies was used to screen for a host response upon mycobacterial infection, reflected as changes in host protein phosphorylation. Changes in the phosphorylation state of 31 known signaling molecules were tracked after infection with live or heat killed Mycobacterium bovis BCG or after incubation with the mycobacterial cell wall component lipoarabinomannan (LAM). Mycobacterial infection triggers a signaling cascade leading to activation of stress-activated protein kinase and its subsequent downstream target, c-Jun. Mycobacteria were also shown to inhibit the activation of protein kinase C epsilon and to induce phosphorylation of proteins not yet known to be involved in mycobacterial infection, such as the cytoskeletal protein alpha-adducin, glycogen synthase kinase 3beta, and a receptor subunit involved in regulation of intracellular Ca(2+) levels. The mycobacterial cell wall component LAM has been identified as a trigger for some of these modulation events.

Antibody Specificity↗

PubMed2004

Zooming in: fractionation strategies in proteomics.

The recent development of mass spectrometry, i.e., high sensitivity, automation of protein identification and some post-translational modifications (PTMs) significantly increased the number of large-scale proteomics projects. However, there are still considerable limitations as none of the currently available proteomics techniques allows the analysis of an entire proteome in a single step procedure. On the other hand, there are several successful studies analyzing well defined groups of proteins, e.g., proteins of purified organelles, membrane microdomains or isolated proteins with certain PTMs. Coupling of advanced separation methodologies (different prefractionation strategies, such as subcellular fractionation, affinity purification, fractionation of proteins and peptides according to their physicochemical properties) to highly sensitive mass spectrometers provides powerful means to detect and analyze dynamic changes of low abundant regulatory proteins in eukaryotic cells on the subcellular level. This review summarizes and discusses recent strategies in proteomics approaches where different fractionation strategies were successfully applied.

Animals↗

PubMed2002

Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation.

BACKGROUND: Proteomic techniques have recently become available for large-scale protein analysis. The utility of these techniques in identification of urinary proteins is poorly defined. We constructed a proteome map of normal human urine as a reference protein database by using two differential fractionated techniques to isolate the proteins. METHODS: Proteins were isolated from urine obtained from normal human volunteers by acetone precipitation or ultracentrifugation, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprinting. RESULTS: A total of 67 protein forms of 47 unique proteins were identified, including transporters, adhesion molecules, complement, chaperones, receptors, enzymes, serpins, cell signaling proteins and matrix proteins. Acetone precipitated more acidic and hydrophilic proteins, whereas ultracentrifugation fractionated more basic, hydrophobic, and membrane proteins. Bioinformatic analysis predicted glycosylation to be the most common explanation for multiple forms of the same protein. CONCLUSIONS: Combining two differential isolation techniques magnified protein identification from human urine. Proteomic analysis of urinary proteins is a promising tool to study renal physiology and pathophysiology and to determine biomarkers of renal disease.

Acetone↗

PubMed2004

Development of high throughput dispersive LC-ion mobility-TOFMS techniques for analysing the human plasma proteome.

A technique that combines ion mobility spectrometry (IMS) with reversed-phase liquid chromatography (LC), collision-induced dissociation (CID) and mass spectrometry (MS) has been developed. The approach is described as a high throughput means of analysing complex mixtures of peptides that arise from enzymatic digestion of protein mixtures. In this approach, peptides are separated by LC and, as they elute from the column, they are introduced into the gas phase and ionised by electrospray ionisation. The beam of ions is accumulated in an ion trap and then the concentrated ion packet is injected into a drift tube where the ions are separated again in the gas phase by IMS, a technique that differentiates ions based on their mobilities through a buffer gas. As ions exit the drift tube, they can be subjected to collisional activation to produce fragments prior to being introduced into a mass spectrometer for detection. The IMS separation can be carried out in only a few milliseconds and offers a number of advantages compared with LC-MS alone. An example of a single 21-minute LC-IMS-(CID)-MS analysis of the human plasma proteome reveals approximately 20,000 parent ions and approximately 600,000 fragment ions and evidence for 227 unique protein assignments.

Chromatography, Liquid↗

PubMed2005

Proteomics and physiology of erythritol-producing strains.

In-depth knowledge bases on physiological properties of microbes are required to design a better microbial system at a gene level and to develop an industrially viable process in an optimized scheme. Proteomic analyses of industrially useful microorganisms are particularly important for achieving such objectives. In this review, industrial application of erythritol in food and pharmaceutical areas and proteomic techniques for erythritol-producing microbes were presented. Proteomic technologies for erythritol-producing strains such as Candida magnoliae contained protein or peptide sample preparation for two-dimensional electrophoresis and mass spectrometry, analysis of proteome with matrix assisted laser desorption-ionization/time-of-flight mass spectrometry, liquid chromatography/electrospray ionization/tandem mass spectrometry and similarity searching algorithms. The proteomic information was applied to predict the carbon metabolism of erythritol-synthesizing microorganisms.

Candida↗

PubMedDate not supplied

Application of proteomic strategies to the identification of urinary biomarkers for prostate cancer: a review.

In the post-genomic era, genes and proteins are now studied on a more comprehensive scale. Studying disease processes at only the genetic or transcriptomic level will give an incomplete amount of information. A proteomic approach potentially allows for a more global overview of how disease processes affect the proteins present in cells, tissues and organisms. The challenge arises in determining which proteins are affected in specific diseases and establishing which of these changes are unique to a particular disease. Existing and emerging proteomic technologies allow for high throughput analysis of proteins in a variety of sample types. Prostate cancer is a significant male health problem in the Western world. It is widely accepted that more specific prognostic and diagnostic markers of prostate cancer are urgently required. The present paper suggests that urine may be an attractive biofluid in which to pursue the identification of novel biomarkers of prostate cancer. This review introduces some proteomic techniques including mass spectrometry and the newer, quantitative proteomic strategies. It focuses on the potential application of these platforms to novel urinary biomarker identification in prostate malignancy. It also includes a synopsis of the current literature on urinary proteomics.

Biomarkers, Tumor↗

PubMed2006

Protein turnover on the scale of the proteome.

Protein turnover is a neglected dimension in postgenomic studies, defining the dynamics of changes in protein expression and forging a link between transcriptome, proteome and metabolome. Recent advances in postgenomic technologies have led to the development of new proteomic techniques to measure protein turnover on a proteome-wide scale. These methods are driven by stable isotope metabolic labeling of cells in culture or in intact animals. This review considers the merits and difficulties of different methods that allow access to proteome dynamics.

Animals↗

PubMed2006

Quantitative proteomic and genomic profiling reveals metastasis-related protein expression patterns in gastric cancer cells.

Gastric cancer is a leading cause of death worldwide, and patients have an overall 5-year survival rate of less than 10%. Using quantitative proteomic techniques together with microarray chips, we have established comprehensive proteome and transcriptome profiles of the metastatic gastric cancer TMC-1 cells and the noninvasive gastric cancer SC-M1 cell. Our qualitative protein profiling strategy offers the first comprehensive analysis of the gastric cancer cell proteome, identifying 926 and 909 proteins from SC-M1 and TMC-1 cells, respectively. Cleavable isotope-coded affinity tagging analysis allows quantitation of a total of 559 proteins (with a protein false-positive rate of <0.005), and 240 proteins were differentially expressed (>1.3-fold) between the SC-M1 and TMC-1 cells. We identified numerous proteins not previously associated with gastric cancer. Notably, a large subset of differentially expressed proteins was associated with tumor metastasis, including proteins functioning in cell-cell and cell-extracellular matrix (cell-ECM) adhesion, cell motility, proliferation, and tumor immunity. Gene expression profiling by DNA microarray revealed differential expression (of >2-fold) of about 1000 genes. The weak correlation observed between protein and mRNA profiles highlights the important complementarities of DNA microarray and proteomics approaches. These comparative data enabled us to map the disease-perturbed cell-cell and cell-ECM adhesion and Rho GTPase-mediated cytoskeletal pathways. Further validation of a subset of genes suggests the potential use of vimentin and galectin 1 as markers for metastasis. We demonstrate that combining proteomic and genomic approaches not only provides a rapid, robust, and sensitive platform to elucidate the molecular mechanisms underlying gastric cancer metastasis but also may identify candidate diagnostic markers and therapeutic targets.

Biomarkers, Tumor↗

PubMed2006

Neuroproteomics of the synapse and drug addiction.

Proteomic analyses of brain tissues are becoming an integral component of neuroscientific research. In particular, the essential role of the synapse in neurotransmission and plasticity has brought about extensive efforts to identify its protein constituents. Recent studies have used a combination of subcellular fractionation and proteomic techniques to identify proteins associated with different components of the synapse. Thus, a coherent map of the synapse proteome is rapidly emerging, and a timely review of these data is warranted. In the first part of this review, neuroproteomic techniques that have been used to analyze the synapse proteome are described. We then summarize the results from several recent proteomic analyses of mammalian synapses and discuss the similarities and differences in their profiling of synaptic proteins. Important advances in this field of research include the use of proteomics to analyze synaptic function and drug effects on synaptic proteins. This article presents an overview of proteomic analyses of the phosphorylation states of synaptic proteins and recent applications of neuroproteomic techniques to the study of drug addiction. Finally, we discuss the challenges in comparing proteomic studies of drug addiction and the future directions of this field in furthering our understanding of the molecular mechanisms underlying synaptic function and drug addiction.

Animals↗

PubMed2005

Asthma, the ugly duckling of lung disease proteomics?

The human respiratory system represents a vital but vulnerable system. It is a major target for many diseases such as cancer and asthma. The incidence of these diseases has increased dramatically in the last 40-50 years. In the search for possible new therapies, many experimental tools and methods have been developed to study these diseases, ranging from animal models to in vitro studies. In the last decades, genomic and proteomic approaches have gained a lot of attention. After the major scientific breakthroughs in the field of genomics, it is now widely accepted that to understand biological processes, large-scale protein studies through proteomics techniques are required. In the battle against lung cancer, the proteomics approach has already been successfully implemented. Surprisingly, only a few proteomics studies on the ever-increasing global asthma problem have been published so far. And although proteomics also has its limitations and experimental difficulties, in our opinion, proteomics can definitely contribute to the understanding of a complex disease such as asthma. Therefore, the additional values and possibilities of proteomics in asthma research should be thoroughly investigated. A close collaboration between the different scientific disciplines may eventually lead to the development of new therapeutic strategies against asthma.

Asthma↗

PubMed2025

T cell population size control by coronin 1 uncovered: from a spot identified by two-dimensional gel electrophoresis to quantitative proteomics.

INTRODUCTION: Recent work identified members of the evolutionarily conserved coronin protein family as key regulators of cell population size. This work originated ~25 years ago through the identification, by two-dimensional gel electrophoresis, of coronin 1 as a host protein involved in the virulence of Mycobacterium tuberculosis. We here describe the journey from a spot on a 2D gel to the recent realization that coronin proteins represent key controllers of eukaryotic cell population sizes, using ever more sophisticated proteomic techniques. AREAS COVERED: We discuss the value of 'old school' proteomics using relatively simple and cost-effective technologies that allowed to gain insights into subcellular proteomes and describe how label-free quantitative (phospho)proteomics using mass spectrometry allowed to disentangle the role for coronin 1 in eukaryotic cell population size control. Finally, we mention potential implications of coronin-mediated cell population size control for health and disease. EXPERT OPINION: Proteome analysis has been revolutionized by the advent of modern-day mass spectrometers and is indispensable for a better understanding of biology. Here, we discuss how careful dissection of physio-pathological processes by a combination of proteomics, genomics, biochemistry and cell biology may allow to zoom in on the unexplored, thereby possibly tackling hitherto unasked questions and defining novel mechanisms.

Proteomics↗

PubMed2006

Proteomic analysis effects of ginsenoside Rg1 on human umbilical vein endothelial cells stimulated by tumor necrosis factor-alpha.

Ginsenoside Rg1 (derived from ginseng root) has been found to have many vasoprotective activities. The present study was undertaken to examine effect of ginsenoside Rg1 on the secretion of nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs) stimulated with or without tumor necrosis factor-alpha (TNF-alpha). We showed here that ginsenoside Rg1 can increase the basal and TNF-alpha-attenuated NO production in a dose-dependent manner. As little is known regarding the vascular molecular mechanism of ginsenoside Rg1 on HUVECs and proteomic technique has more advantages in molecular identification, we attempted to use proteomic analysis to explain vascular molecular mechanism of ginsenoside Rg1 on HUVECs. Proteomic analytical result showed that 21 protein spots were changed in TNF-alpha stimulated HUVECs, including 9 up-regulated spots, 11 down-regulated spots, and 1 spot detected in TNF-alpha stimulated group only. The expression level of proteins such as MEKK3, phosphoglycerate mutase was increased, and nitric-oxide synthase, mineralocorticoid receptor were decreased in TNF-alpha stimulated HUVECs, while ginsenoside Rg1 could prevent this change or reverse to some degree. This study suggested that NO production increased via ginsenoside Rg1 played an important role in the protective effect on TNF-alpha stimulated HUVECs and was helpful to deeply understand the active mechanism of ginsenoside Rg1 to HUVECs at the molecular level.

Blotting, Western↗

PubMed2005

Proteomic determination of widespread detergent-insolubility including Abeta but not tau early in the pathogenesis of Alzheimer's disease.

Biochemical characterization of the major detergent-insoluble proteins that comprise hallmark histopathologic lesions initiated the molecular era of Alzheimer's disease (AD) research. Here, we reinvestigated detergent-insoluble proteins in AD using modern proteomic techniques. Using liquid chromatography (LC)-mass spectrometry (MS)-MS-based proteomics, we robustly identified 125 proteins in the detergent-insoluble fraction of late-onset AD (LOAD) temporal cortex that included several proteins critical to Abeta production, components of synaptic scaffolding, and products of genes linked to an increased risk of LOAD; we verified 15 of 15 of these proteins by Western blot. Following multiple analyses, we estimated that these represent ~80% of detergent-insoluble proteins in LOAD detectable by our method. Abeta, tau, and 7 of 8 other newly identified detergent-insoluble proteins were disproportionately increased in temporal cortex from patients with LOAD and AD derived from mutations in PSEN1 and PSEN2; all of these except tau were elevated in individuals with prodromal dementia, while none except Abeta were elevated in aged APPswe mice. These results are consistent with the amyloid hypothesis of AD and extend it to include widespread protein insolubility, not exclusively Abeta insolubility, early in AD pathogenesis even before the onset of clinical dementia.

Alzheimer Disease↗

PubMed2002

Proteomic study of non-typable Haemophilus influenzae.

Non-typable Haemophilus influenzae (NTHi) are small, gram-negative bacteria and are strictly human pathogens, causing acute otitis media, sinusitis and community-acquired pneumonia. There is no vaccine available for NTHi, as there is for H. influenzae type b. Recent advances in proteomic techniques are finding novel applications in the field of vaccinology. There are several protein separation techniques available today, each with inherent advantages and disadvantages. We employed a combined proteomics approach, including sequential extraction and analytical two-dimensional polyacrylamide electrophoresis (2D PAGE), and two-dimensional semi-preparative electrophoresis (2D PE), in order to study protein expression in the A4 NTHi strain. Although putative vaccine candidates were identified with both techniques, 11 of 15 proteins identified using the 2D PE approach were not identified by 2D PAGE, demonstrating the complementarily of the two methods.

Bacterial Proteins↗

PubMed2005

Proteomics in prostate cancer.

PURPOSE OF REVIEW: State-of-the-art proteomics technologies are currently being assessed for utility in the study of prostatic malignancy. This review aims to provide background information on the current proteomics techniques employed in prostate cancer research, recent reports showing the potential application of proteomics in urological practice, and the future direction of proteomics in prostate cancer research and management. RECENT FINDINGS: Proteomic profiling of serum as a diagnostic tool and a platform for biomarker discovery in prostate cancer continues to draw favorable attention as well as close scrutiny as technological enhancements and multi-center study results are reported. In-vitro studies on prostate cell lines provide positive proof-of-principle results. The application of proteomics to query prostate tissue specimens yields novel prostate cancer biomarkers requiring further validation. The integration of proteomics with immunology also yields promising findings that may translate into clinically relevant biological assays. SUMMARY: The study of proteomics is an emerging research field, and current studies continue to display potential future usage in prostate cancer management. Succeeding scientific investigations will probably yield new diagnostic and prognostic tools for prostate cancer, provide insights into its underlying biology, and contribute to the development of novel treatment strategies.

Gene Expression↗

PubMed2005

Functional Genomics meets neurodegenerative disorders. Part II: application and data integration.

The transcriptomic and proteomic techniques presented in part I (Functional Genomics meets neurodegenerative disorders. Part I: transcriptomic and proteomic technology) of this back-to-back review have been applied to a range of neurodegenerative disorders, including Huntington's disease (HD), Prion diseases (PrD), Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), frontotemporal dementia (FTD) and Parkinson's disease (PD). Samples have been derived either from human brain and cerebrospinal fluid, tissue culture cells or brains and spinal cord of experimental animal models. With the availability of huge data sets it will firstly be a major challenge to extract meaningful information and secondly, not to obtain contradicting results when data are collected in parallel from the same source of biological specimen using different techniques. Reliability of the data highly depends on proper normalization and validation both of which are discussed together with an outlook on developments that can be anticipated in the future and are expected to fuel the field. The new insight undoubtedly will lead to a redefinition and subdivision of disease entities based on biochemical criteria rather than the clinical presentation. This will have important implications for treatment strategies.

Animals↗

PubMed2006

Activity-based proteomics: enzymatic activity profiling in complex proteomes.

In the postgenomic era new technologies are emerging for global analysis of protein function. The introduction of active site-directed chemical probes for enzymatic activity profiling in complex mixtures, known as activity-based proteomics has greatly accelerated functional annotation of proteins. Here we review probe design for different enzyme classes including serine hydrolases, cysteine proteases, tyrosine phosphatases, glycosidases, and others. These probes are usually detected by their fluorescent, radioactive or affinity tags and their protein targets are analyzed using established proteomics techniques. Recent developments, such as the design of probes for in vivo analysis of proteomes, as well as microarray technologies for higher throughput screenings of protein specificity and the application of activity-based probes for drug screening are highlighted. We focus on biological applications of activity-based probes for target and inhibitor discovery and discuss challenges for future development of this field.

Animals↗