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PubMedDate not supplied

A review of proteomics in cancer research.

The most functional compartment encoded by the genome is proteome. Therefore study of proteome i.e. proteomics is the promising approach in identification, separation and quantitation of functional changes. It aims to gain a comprehensive understanding of the expressions, modifications, interactions, and regulation of proteins in cells. The power of two-dimensional electrophoresis and advances in mass spectrometric techniques, combined with sequence data base correlation, has enabled speed and accuracy in identification of proteins in complex mixtures. Therefore, proteomics may provide a better understanding of the molecular basis of cancer growth, with identification of potential pathological markers and therapeutic targets. Tobacco related cancers are the major health hazard in Asian countries; the proteomics approach should be employed for understanding the underlying disease processes and hopefully reveal important clues for identifying high risk individuals and early changes during malignant transformation.

Biomarkers, Tumor↗
PubMed2026

A porcine spectral assay library to quantify brain proteome by DIA-MS.

Neurological disorders are the leading cause of health loss worldwide. The growing number of patients suffering from such conditions calls for improved strategies for their prevention, diagnosis, and therapy. To better understand human pathologies, relevant models and methodologies must be made available. In this study, we focused on a biomedical model capable of recapitulating the complexity of human pathology, the pig (Sus scrofa). Brain tissue and cerebrospinal fluid samples from a transgenic minipig model of Huntington's disease were subjected to multiple extraction and fractionation steps. A proteomic mass spectrometry (MS) methodology then allowed the generation of a porcine spectral library for 8,321 proteins. Using data-independent acquisition (DIA), we demonstrated that our porcine spectral library substantially enhanced the quantitative potential of this untargeted MS approach, generating reproducible proteome-wide data. The porcine library also provides a comprehensive resource for the development of targeted MS assays, enabling the quantification of selected proteins with a key role not only in neuroscience.

Animals↗
PubMed2026

Acidic Stress Induces Proteomic Reprogramming and Virulence-Associated Adaptation in Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis whose etiologic agents must adapt to acidic host niches such as phagolysosomes. Here, we used quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics to define the response of Paracoccidioides brasiliensis to acidic stress (pH 4.5) versus control pH (6.5) after 5 and 24 h. We identified and quantified 4374 proteins, including 327 and 722 differentially abundant proteins at 5 and 24 h, respectively, revealing time-dependent proteomic reprogramming. Enrichment analyses highlighted proteolysis, protein metabolism, organonitrogen metabolism, MAPK- and SNF1-like signaling, central carbon metabolism, tyrosine metabolism, and ergosterol biosynthesis as major acid-responsive processes. Complementary assays showed pH-dependent extracellular proteolytic activity, increased adhesion to A549 pulmonary epithelial cells, and dynamic ergosterol remodeling. The proteomic data further indicated increased abundance of moonlighting proteins linked to adhesion and metabolic enzymes associated with ATP generation and melanin precursor production. Together, these findings indicate that P. brasiliensis adapts to acidic environments through coordinated regulation of proteostasis, metabolism, signaling, host-cell interaction, and membrane homeostasis, supporting survival and virulence potential in acidic host microenvironments.

Paracoccidioides↗
PubMed2006

Simultaneous proteomic profiling of four different growth states of human fibroblasts, using amine-reactive isobaric tagging reagents and tandem mass spectrometry.

In general, permanent growth arrest due to exhaustive cell replication can be induced prematurely by either stress or overexpression of selected oncogenes. In an attempt to examine key proteins involved in achieving premature senescence, and how they differ from those in serially passaged, replicatively exhausted cells, we used a novel proteomic profiling approach, isobaric tagging for relative and absolute quantitation (iTRAQ), to perform simultaneous four-way comparison of replicatively senescent fibroblasts, oxidatively stressed prematurely senescent fibroblasts, and their young replicating and quiescent counterparts. Two hundred and forty proteins were identified and quantified simultaneously; data analysis reveals: (1) groups of proteins whose expressions are uniformly either up- or down-regulated in all three growth arrest states; (2) signature proteins which may serve as candidate proteomic markers to differentiate the quiescent state from permanent growth arrest by either exhaustive replication or stress induction and (3) that while oxidative stress-induced, prematurely senescent fibroblasts morphologically resemble their replicatively exhausted counterparts, they exhibit different protein expression patterns. Results from simultaneous proteomic profiling were validated by Western blotting for selected proteins: collagen type I, HSP90 and vimentin. In conclusion, this report shows that iTRAQ proteomic profiling is a powerful technique for globally mapping protein signatures for different culture growth states.

Amino Acid Sequence↗
PubMed2003

Protein microarrays: molecular profiling technologies for clinical specimens.

Proteomics, the study of protein function within biologic systems, will further our understanding of cancer pathogenesis. Coupled with transcript profiling, proteomics can herald the advent of molecular therapy tailored to the individual patient's neoplasm. Protein microarrays, one emerging class of proteomic technologies, have broad applications for discovery and quantitative analysis. This technology is uniquely suited to gather information about the post-translational modifications of proteins reflecting the activity state of signal pathways and networks. Protein microarrays now make it feasible to conduct signal network profiling within cellular samples. Nevertheless, to be successful, design and use of protein microarrays must take into consideration enormous analytical challenges. A subclass of protein microarrays, Reverse Phase Arrays, created to meet these challenges, has been optimized for use with tissue specimens, and is now in use for the analysis of biopsy samples for clinical trial research.

Antibodies↗
PubMed2003

A new quantitative optical biosensor for protein characterisation.

A new optical biosensor is described based on a dual waveguide interferometric technique. By addressing the waveguide structure with alternate polarisations the optogeometrical properties (density and thickness) of adsorbed protein layers at the sensor (solid)-liquid interface have been determined. Differences in the waveguide mode dispersion between the transverse electric (TE) and transverse magnetic (TM) modes allow unique solutions for adlayer thickness and refractive index to be determined at all stages during the formation process. The technique has been verified using standard protein systems and by comparing the data with published work using X-ray crystallography and neutron reflection techniques. The data obtained was found to be in excellent agreement with previously reported X-ray experiments given that typical film thicknesses for streptavidin layers were in the range 5.5-6.5 nm compared with the short axis crystal structure of between 4.8 and 5.6 nm. The precision of the measurements taken was of the order of 40 pm with respect to adsorbed adlayer thicknesses. This biosensor approach provides measurements of both thickness and density of adlayers to a high precision, simultaneously and in real time enabling detail of the structure and function of proteins to be elucidated. From such data it is possible to obtain information on the orientation, distortion and efficiency of immobilisation procedures as well as the interaction event of interest. The technique is expected to find utility with those interested in protein structure and function. This is an area of growing importance within the life sciences as the demand for quantitative analytical techniques increases with the growth in "proteomics".

Antibodies↗
PubMed2003

Molecular diagnostics in medical microbiology: yesterday, today and tomorrow.

Clinical microbiology is clearly on the move, and various new diagnostic technologies have been introduced into laboratory practice over the past few decades. However, Henri D Isenberg recently stated that molecular biology techniques promised to revolutionise the diagnosis of infectious disease, but that, to date, this promise is still in its infancy. Molecular diagnostics have now surpassed these early stages and have definitely reached puberty. Currently, a second generation of automated molecular approaches is already within the microbiologists' reach. Quantitative amplification tests in combination with genomics, transcriptomics, proteomics and related methodologies will pave the way to further enhancement of innovative microbial detection and identification.

Genomics↗
PubMedDate not supplied

Application of isotope coded affinity tag (ICAT) analysis for the identification of differentially expressed proteins following infection of atlantic salmon (Salmo salar) with infectious hematopoietic necrosis virus (IHNV) or Renibacterium salmoninarum (BKD).

Aquaculture and commercial fisheries worldwide suffer from significant economic loss due to diseases of net-pen reared fish. In British Columbia, infectious hematopoietic necrosis (IHN) and bacterial kidney disease (BKD) epidemics occur because there are currently no commercially available drugs or fully licensed vaccines to treat these diseases. With a better understanding of the molecular mechanisms underlying these diseases, this circumstance might be significantly improved. In the present study, we have used a proteomics approach in an effort to identify and quantitate differentially expressed proteins in the liver and kidneys of diseased and healthy Atlantic salmon (Salmo salar). Isotope coded affinity tagging (ICAT), 2D gel electrophoresis, and multidimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC MS/MS) were used to identify hundreds of differentially expressed proteins. While the direct significance of changes in expression levels of many proteins remains to be elucidated, others appear to be more clearly related to the infectious process. Examples of the latter are discussed here and include, a natural killer cell enhancement factor (NKEF), procathepsin L, superoxide-producing NADPH oxidase and interferon-induced viral resistance protein Mx (IFI-Mx).

Affinity Labels↗
PubMed2006

Protein chip discovery of secreted proteins regulated by the phosphatidylinositol 3-kinase pathway in ovarian cancer cell lines.

Ovarian cancer has the highest mortality among the gynecologic malignancies. The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated, leading to increased cell survival. This study aimed to identify secreted proteins regulated by the PI3K pathway in ovarian cancer cell lines. Surface-enhanced laser desorption-ionization time-of-flight mass spectrometry with cation-exchange protein-chips was used to analyze secreted proteins from five ovarian cancer cell lines (SKOV-3, PE01, OVCAR-3, OV167, and OV207). To activate the PI3K pathway, cells were treated with 50 ng/mL epidermal growth factor (EGF) with or without 10 micromol/L LY294002, a PI3K inhibitor. Proteins induced by EGF and inhibited by LY294002, in the m/z range 7,500 to 9,500, were purified chromatographically, identified by peptide mass fingerprinting and NH(2)-terminal sequencing, and confirmed by immunodepletion. Two immunologically related proteins, m/z approximately 8,385 and 8,922, were identified as truncated and intact forms, respectively, of interleukin 8, a chemokine previously shown to be elevated in serum of ovarian cancer patients. Another protein, m/z 7,866, was identified as CXC chemokine ligand 1 (CXCL1) or GRO-alpha, a chemokine associated with melanoma formation and some epithelial cancers. EGF-stimulated CXCL1 levels were variably decreased by mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase and p38 MAPK inhibition in the five cell lines, but only LY294002 fully reversed the EGF effect in all cell lines. Immunoreactive CXCL1 levels in 160 conditioned media were highly correlated with corresponding peak intensities at m/z 7,866 by mass spectrometry, indicating the quantitative nature of these analyses. We conclude that proteomic analysis of cell models of human disease may facilitate the discovery of pathway-dependent proteins.

Blotting, Western↗
PubMed2003

The Database of Quantitative Cellular Signaling: management and analysis of chemical kinetic models of signaling networks.

MOTIVATION: Analysis of cellular signaling interactions is expected to pose an enormous informatics challenge, perhaps even larger than analyzing the genome. The complex networks arising from signaling processes are traditionally represented as block diagrams. A key step in the evolution toward a more quantitative understanding of signaling is to explicitly specify the kinetics of all chemical reaction steps in a pathway. Technical advances in proteomics and high-throughput protein interaction assays promise a flood of such quantitative data. While annotations, molecular information and pathway connectivity have been compiled in several databases, and there are several proposals for general cell model description languages, there is currently little experience with databases of chemical kinetics and reaction level models of signaling networks. RESULTS: The Database of Quantitative Cellular Signaling is a repository of models of signaling pathways. It is intended both to serve the growing field of chemical-reaction level simulation of signaling networks, and to anticipate issues in large-scale data management for signaling chemistry. AVAILABILITY: The Database of Quantitative Cellular Signaling is available at http://doqcs.ncbs.res.in. Links to the signaling model simulator, GENESIS/Kinetikit are at http://www.ncbs.res.in/~bhalla/kkit/index.html and are also provided from within the database. The database source code is available under the GNU Public License.

Abstracting and Indexing↗
PubMed2004

Proteome analysis of DNA damage-induced neuronal death using high throughput mass spectrometry.

Isotope-coded affinity tag reagents and high throughput mass spectrometry were used to quantitate changes in the expression of 150 proteins in mouse wild-type (p53(+/+)) cortical neurons undergoing DNA damage-induced death. Immunological techniques confirmed several of the changes in protein expression, but microarray analysis indicated that many of these changes were not accompanied by altered mRNA expression. Proteome analysis revealed perturbations in mitochondrial function, free radical production, and neuritogenesis that were not observed in p53-deficient neurons. Changes in Tau, cofilin, and other proteins recapitulated abnormalities observed in neurodegenerative states in vivo. Additionally, DNA damage caused a p53-dependent decrease in expression of members of the protein kinase A (PKA) signaling pathway. PKA inhibition promoted death in the absence of DNA damage, revealing a novel mechanism by which endogenous down-regulation of PKA signaling may contribute to p53-dependent neuronal death. These data demonstrate the power of high throughput mass spectrometry for quantitative analysis of the neuronal proteome.

Animals↗
PubMed2006

Serum proteomic signature for cystic fibrosis using an antibody microarray platform.

Antibody microarrays are a new proteomic technology, which we have developed as a platform for identifying a cystic fibrosis (CF)-specific serum proteomic signature. Serum samples from CF patients have been pooled and compared with equivalent pools of control sera in order to identify patterns of protein expression unique to CF. We find that the set of significantly differentially expressed proteins is enriched in protein mediators of inflammation from the NFkappaB signaling pathway, and in proteins that may be selectively expressed in CF-affected tissues such as lung and intestine. In several instances, we validate the data from the antibody microarrays by quantitative analysis with Reverse Capture Protein Microarrays. We conclude that antibody microarray technology is sensitive, quantitative, and robust, and can be useful as a proteomic platform to discriminate between sera from CF and control patients.

Adolescent↗
PubMed2006

Proteomics analysis of the actions of grape seed extract in rat brain: technological and biological implications for the study of the actions of psychoactive compounds.

Grape seed extract (GSE) is a commonly available dietary supplement taken for the anti-oxidant activity that's attributed to its proanthocyanidin (oligomers of monomeric polyphenols) content. Similar polyphenol-enriched preparations from blueberries and soy have shown protection against ovariectomy-induced or age-related cognitive deficits, suggesting that the molecular changes induced by these polyphenol preparations correlated with behavioral benefit. We hypothesized that ingestion of polyphenol-enriched preparations such as GSE would be manifested as protein changes that would be consistent with neuroprotection. Proteomics technology, namely 2D gel electrophoresis and mass spectrometry, identified quantitative changes in specific proteins induced in adult rat brain following ingestion of a powdered preparation of GSE. As recently reported [Deshane, J., Chaves, L., Sarikonda, K.V., Isbell, S., Wilson, L., Kirk, M., Grubbs, C., Barnes, S., Meleth, S. and Kim, H., 2004. Proteomics analysis of rat brain protein modulations by grape seed extract. Journal of Agricultural and Food Chemistry 52, 7872-7883.], the direction of change for the majority of the affected proteins was opposite to the direction the proteins were changed in either Alzheimer disease or in transgenic mouse models of dementia. A conservative conclusion is that GSE has neuroprotective activity, by affecting specific proteins in particular ways. In this chapter, elements of proteomics-type analysis are discussed that demonstrate the power of the technology to enable discovery of proteins involved in the response of the brain to a stimulus whether it be a dietary supplement, or a psychoactive drug. The fact that GSE affects proteins implicated in cognitive disorders suggests moreover that GSE may have impact on the actions of psychoactive drugs by maintaining an overall viability of the nervous system.

Animals↗
PubMed2006

Proteomic applications of protein quantification by isotope-dilution mass spectrometry.

Over the decades, isotope-dilution mass spectrometry (IDMS) has been implemented extensively for accurate quantification of drugs, metabolites and peptides in body fluids and tissues. More recently, it has been extended for quantifying specific proteins in complex mixtures. In this extended methodology, proteins are subjected to endoprotease action and specific resultant peptides are quantified by using synthetic stable isotope-labeled standard (SIS) peptides and IDMS. This article outlines the utilities and applications of quantifying proteins by IDMS, emphasizing its complementary value to global survey-based proteomic studies. The potential of SIS peptides to provide quantitative insights into cell signaling is also highlighted, with specific examples. Finally, we propose several novel mass spectrometric data acquisition strategies for large-scale applications of IDMS and SIS peptides in systems biology and protein biomarker validation studies.

Animals↗
PubMed2006

A proteomic approach for unraveling the oncogenic H-Ras protein networks in NIH/3T3 mouse embryonic fibroblast cells.

To elucidate the oncogenic H-Ras network, we have established various stable and inducible oncogenic H-Ras-expressing NIH/3T3 mouse embryonic fibroblast cell clones, which express G12V H-Ras and G12R H-Ras proteins under the influence of a strong cytomegalovirus promoter and under the tight control of expression by an antibiotic, doxycycline, respectively. Here we provide a catalogue of proteome profiles in total cell lysates derived from oncogenic H-Ras-expressing NIH/3T3 cells. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis and MALDI-TOF MS analysis using both a stable expression system as well as an inducible expression system. There were a large number of common targets for oncogenic H-Ras, which were identified in both cell lines and consisted of 64 proteins (36 up-regulated and 28 down-regulated). Differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. Taken together, the results presented here show that comparative analysis of the proteome from the oncogenic H-Ras-expressing cells yielded interpretable data to elucidate protein networks directly and/or indirectly.

Animals↗
PubMed2006

Decoding 2D-PAGE complex maps: relevance to proteomics.

This review describes two mathematical approaches useful for decoding the complex signal of 2D-PAGE maps of protein mixtures. These methods are helpful for interpreting the large amount of data of each 2D-PAGE map by extracting all the analytical information hidden therein by spot overlapping. Here the basic theory and application to 2D-PAGE maps are reviewed: the means for extracting information from the experimental data and their relevance to proteomics are discussed. One method is based on the quantitative theory of statistical model of peak overlapping (SMO) using the spot experimental data (intensity and spatial coordinates). The second method is based on the study of the 2D-autocovariance function (2D-ACVF) computed on the experimental digitised map. They are two independent methods that are able to extract equal and complementary information from the 2D-PAGE map. Both methods permit to obtain fundamental information on the sample complexity and the separation performance and to single out ordered patterns present in spot positions: the availability of two independent procedures to compute the same separation parameters is a powerful tool to estimate the reliability of the obtained results. The SMO procedure is an unique tool to quantitatively estimate the degree of spot overlapping present in the map, while the 2D-ACVF method is particularly powerful in simply singling out the presence of order in the spot position from the complexity of the whole 2D map, i.e., spot trains. The procedures were validated by extensive numerical computation on computer-generated maps describing experimental 2D-PAGE gels of protein mixtures. Their applicability to real samples was tested on reference maps obtained from literature sources. The review describes the most relevant information for proteomics: sample complexity, separation performance, overlapping extent, identification of spot trains related to post-translational modifications (PTMs).

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Towards validating a method for two-dimensional electrophoresis/silver staining.

Two-dimensional electrophoresis (2-DE) is a technique involving numerous steps, many of them to be performed manually. Hence, some operator dependency must be taken into account. An attempt to elucidate the reliability of 2-DE combined with silver staining is presented, employing the general practice to validate a method in pharmaceutical analysis. Most proteomic studies employing 2-DE aim at qualitative or quantitative differences in protein expression. One of the most sensitive and broadly applied staining techniques is silver staining. In order to gain information on accuracy, precision, linearity, and ruggedness of this technique, gels were run in replicates with different amounts of protein from a complex standard sample. In addition, sets of gels were repeated by two different operators in a second independent laboratory equipped with identical hardware and software. Our results show that reliable qualitative data on differential protein expression can be obtained by 2-DE, nevertheless replicate gels should be run and experimental conditions have to be kept stringently to a standardized protocol. Quantitative data are just achievable with spots, which are well-resolved, of high quality, with an optical density (OD) above a certain threshold (OD > 10), and which show a linear response. Quantitative differences occurring due to method-derived deviations may easily be misinterpreted as true changes in protein expression. After normalization, relative standard deviation (RSD) values of approximately 30% (n = 4) could be obtained, therefore minor changes (< 50%) should be critically reviewed.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Mapping glycosylation changes related to cancer using the Multiplexed Proteomics technology: a protein differential display approach.

The metastatic spread of tumor cells in malignant progression is known to be a major cause of cancer mortality. Protein glycosylation is increasingly being recognized as one of the most prominent biochemical alterations associated with malignant transformation and tumorigenesis. The Multiplexed Proteomics (MP) approach is a new technology that permits quantitative, multicolor fluorescence detection of proteins in two-dimensional (2-D) gels and on Western blots. This methodology allows the parallel determination of both altered glycosylation patterns and protein expression level changes within a single 2-D gel experiment. The linear responses of the fluorescent dyes utilized allow rigorous quantitation of changes in protein expression over a broad 3-log linear dynamic range. Global analysis of changes in protein glycosylation and total protein expression is followed by dichromatic, lectin-based profiling methods for rapidly categorizing glycan branching structures. The MP approach was applied to whole tissue extracts of normal and cancerous liver, so that altered glycosylation modification patterns and protein expression levels could be determined. One prominent glycoprotein determined to be up-regulated in the tumor tissue was haptoglobin, an acute-phase response protein. The detection methodologies associated with the MP technology radically increase the information content of 2-D gel experiments. This new information greatly enhances the applicability of these experiments in addressing fundamental questions associated with proteome-wide glycosylation changes related to cancer.

Electrophoresis, Gel, Two-Dimensional↗