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Results for “Proteomics techniques”

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PubMed2006

N-glycosylation analysis using the StrOligo algorithm.

N-glycosylation of proteins is the predominant glycosylation in mammals and confers specific conformations, localization, and functions to proteins. High-throughput proteomics techniques have focused on the identification of proteins through amino acid sequence determination, with little attention paid to their post-translational modification, in particular, glycosylation. High-throughput mass spectrometric data often contain information about glycosylation, but this is systematically discarded by proteomic search engines. We have developed an algorithm, StrOligo (for STRucture of OLIGOsaccharides), capable of automated analysis of oligosaccharide composition and possible structures by mass spectrometry. The algorithm analyzes tandem mass spectrometry (MS/MS) data in an automated three-step process and provides possible structures and a discrimination score. In the first step, the algorithm constructs a relationship tree of the monosaccharide moiety losses observed in the MS/MS spectrum. In the second step, the algorithm uses the tree to propose possible compositions and structures from combinations of adduct and fragment ions as well as a discrimination score, which reflects the fit with the experimental results. Finally, an interface is available to visualize the proposed structures and their scores. As well, the MS/MS spectrum is displayed with relevant peaks labeled for the proposed structure with the highest discrimination score, using a modified nomenclature.

Algorithms↗

PubMed2006

Neuroproteomics: relevance to anxiety disorders.

Despite advances in the treatment of anxiety disorders, there is a need for medications with greater efficacy and fewer side effects. Advances in techniques to facilitate high throughput, mass analysis of proteins potentially allows for new drug targets, with a shift in focus from membrane receptor proteins and enzymes of neurotransmitter metabolism to molecules in intracellular signal transduction and other pathways. A computerized literature search was done to collect studies on recently developed proteomic techniques and their application in psychiatric research. Particular techniques, such as two-dimensional electrophoresis, two-dimensional differential gel electrophoresis, isotope-coded affinity tags, and isotope tags for relative and absolute quantification, are reviewed. In addition, a combination of these techniques with MALDI-TOF/TOF and ESI-Q-TOF mass spectrometry analysis is discussed in relation to possible novel signaling pathways relevant to anxiety disorders, and to the development of biomarkers for the evaluation of these conditions.

Animals↗

PubMed2006

The proteomics of keratin proteins.

Keratin proteins are widespread in nature, being found in the nuclei and cytoplasm of almost all differentiated eukaryote cells. However, they are best known as the principal structural proteins in hair, wool and skin. Because of difficulties associated with their extraction from biological samples, high sequence homology and the presence of numerous post-translational modifications, they have been less well studied than other protein families. Thanks to the advent of modern proteomic techniques we now have available a good suite of tools to study this neglected family of proteins.

Amino Acid Sequence↗

PubMed2006

Heterogeneous nuclear ribonuclear protein C is increased in the celecoxib-induced growth inhibition of human oral squamous cell carcinoma.

Celecoxib is a selective inhibitor of cyclooxygenase-2 (COX-2) that is a critical factor in carcinogenesis, but precise mechanism of its action remains to be elucidated. Here we evaluated the inhibitory effect of celecoxib on cell growth of human oral squamous cell carcinoma (OSCC) YD-10(B), which was established to be used as in vitro OSCC model, and identified celecoxib-regulated protein by proteomics techniques. Celecoxib (IC(50)=37 microM) inhibited the growth of YD-10(B) cells with the decrease of COX-2 protein expression. Its inhibition could be linked in the arrest of G(1) phase with increased levels of p(27) protein, a specific CDK inhibitor. Using proteomics, the 10- to 20-fold increase of heterogeneous nuclear ribonuclear protein C (hnRNP C), which has been suggested to be related with the translation of p(27) mRNA, was observed in celecoxib-treated YD-10(B) cells. In summary, celecoxib has a potential to induce the protein expression of hnRNP C and its increase subsequently induce the translation of p(27) mRNA, which trigger the inhibition of cell growth via p(27)-regulated cell cycle arrest in YD-10(B) cells. In addition, YD-10(B) cells could be useful to study the pathological mechanism of OSCC.

Actins↗

PubMed2005

Classification and identification of bacteria using mass spectrometry-based proteomics.

Timely classification and identification of bacteria is of vital importance in many areas of public health. Mass spectrometry-based methods provide an attractive alternative to well-established microbiologic procedures. Mass spectrometry methods can be characterized by the relatively high speed of acquiring taxonomically relevant information. Gel-free mass spectrometry proteomics techniques allow for rapid fingerprinting of bacterial proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or, for high-throughput sequencing of peptides from protease-digested cellular proteins, using mass analysis of fragments from collision-induced dissociation of peptide ions. The latter technique uses database searching of product ion mass spectra. A database contains a comprehensive list of protein sequences translated from protein-encoding open reading frames found in bacterial genomes. The results of such searches allow the assignment of experimental peptide sequences to matching theoretical bacterial proteomes. Phylogenetic profiles of sequenced peptides are then used to create a matrix of sequence-to-bacterium assignments, which are analyzed using numerical taxonomy tools. The results thereof reveal the relatedness between bacteria, and allow the taxonomic position of an investigated strain to be inferred.

Animals↗

PubMed2026

Proteomic profiling of bone for the estimation of post-mortem interval and post-mortem submersion interval: a systematic review.

Accurate estimation of the Post-Mortem Interval (PMI) and Post-Mortem Submersion Interval (PMSI) remains a persistent challenge in forensic science, especially when traditional morphological and entomological methods fail due to advanced decomposition or in aquatic environments. Proteomic profiling of bone tissues has recently emerged as a promising approach, leveraging the predictable degradation patterns of bone proteins to estimate time since death more reliably. This systematic review, conducted in accordance with PRISMA guidelines, analyzed 24 peer-reviewed studies focusing on the application of proteomic techniques to bone tissue for PMI and PMSI estimation. The included studies were evaluated based on sample type, analytical techniques used, identified biomarkers, environmental conditions assessed, and the overall reliability and reproducibility of the findings. The review found that specific bone proteins, particularly collagen, osteocalcin, fetuin-A, etc. exhibited consistent degradation patterns that correlated strongly with elapsed post-mortem time. Cortical bone was identified as a more stable and informative matrix compared to trabecular bone. Mass spectrometry, especially LC-MS/MS, emerged as the predominant analytical technique due to its high sensitivity and accuracy in detecting low-abundance proteins over extended PMIs and PMSIs. However, protein degradation rates were significantly influenced by environmental variables such as temperature, humidity, soil pH, and microbial activity. This review also emphasizes the transformative role of bone proteomics in advancing forensic science while identifying key gaps that must be addressed to achieve global standardization and practical implementation in diverse forensic contexts. The integration of proteomics with other emerging technologies, such as machine learning algorithms and computational modeling, may further enhance the precision of PMI and PMSI estimation in future applications.

Postmortem Changes↗

PubMed2004

Oral flora in the age of molecular biology.

The present work describes the fundamental aspects of the molecular methods that are applied to oral microbiology: probes, PCR, multiple real time PCR, 16S rDNA, and proteomics. Likewise, it presents the results obtained by applying these methods to the study of the bacterial flora encountered inside the mouth. By identifying 16S rDNA, up to 132 known species and 215 new, unknown phylotypes have been detected inside patients s periodontal pockets. We are currently using a commercial system based on multiple PCR (genomic amplification), followed by reverse hybridization to detect the five species known to cause periodontal disease. We also summarize the findings derived from the application of proteomic techniques to the study of odontologic infections pathogenesis and from the use of molecular methods in the study of resistance to antimicrobial agents. Finally, it puts forth the problems that remain unsolved with respect to oral flora and the treatment of odontogenic infections. Traditional culture methods continue to be indispensable, as they make it possible to later work with the cultured microorganisms.

Aggregatibacter actinomycetemcomitans↗

PubMed2003

Toxicogenomic approach for assessing toxicant-related disease.

The problems of identifying environmental factors involved in the etiology of human disease and performing safety and risk assessments of drugs and chemicals have long been formidable issues. Three principal components for predicting potential human health risks are: (1) the diverse structure and properties of thousands of chemicals and other stressors in the environment; (2) the time and dose parameters that define the relationship between exposure and disease; and (3) the genetic diversity of organisms used as surrogates to determine adverse chemical effects. The global techniques evolving from successful genomics efforts are providing new exciting tools with which to address these intractable problems of environmental health and toxicology. In order to exploit the scientific opportunities, the National Institute of Environmental Health Sciences has created the National Center for Toxicogenomics (NCT). The primary mission of the NCT is to use gene expression technology, proteomics and metabolite profiling to create a reference knowledge base that will allow scientists to understand mechanisms of toxicity and to be able to predict the potential toxicity of new chemical entities and drugs. A principal scientific objective underpinning the use of microarray analysis of chemical exposures is to demonstrate the utility of signature profiling of the action of drugs or chemicals and to utilize microarray methodologies to determine biomarkers of exposure and potential adverse effects. The initial approach of the NCT is to utilize proof-of-principle experiments in an effort to "phenotypically anchor" the altered patterns of gene expression to conventional parameters of toxicity and to define dose and time relationships in which the expression of such signature genes may precede the development of overt toxicity. The microarray approach is used in conjunction with proteomic techniques to identify specific proteins that may serve as signature biomarkers. The longer-range goal of these efforts is to develop a reference relational database of chemical effects in biological systems (CEBS) that can be used to define common mechanisms of toxicity, chemical and drug actions, to define cellular pathways of response, injury and, ultimately, disease. In order to implement this strategy, the NCT has created a consortium of research organizations and private sector companies to actively collaborative in populating the database with high quality primary data. The evolution of discrete databases to a knowledge base of toxicogenomics will be accomplished through establishing relational interfaces with other sources of information on the structure and activity of chemicals such as that of the National Toxicology Program (NTP) and with databases annotating gene identity, sequence, and function.

Animals↗

PubMed2003

Application of proteomics to the study of molecular mechanisms in neurotoxicology.

The proteome is the protein compliment of the genome and is the result of genetic expression, ribosomal synthesis and proteolytic degradation. Proteins participate in most major cell processes and their function is highly regulated by post-translational modifications such as phosphorylation and glycosylation. As a result, neurotoxicant-induced changes in protein levels, function or regulation could have a negative impact on neuronal viability. At the molecular level, direct oxidative or covalent modifications of individual proteins by various chemicals or drugs is likely to lead to perturbation of tertiary structure and a loss of function. The proteome and the functional determinants of its individual protein components are, therefore, likely targets of neurotoxicant action and resulting characteristic disruptions could be critically involved in corresponding mechanisms of neurotoxicity. Clearly, investigating changes in the proteome can provide important clues for deciphering mechanisms of toxicant action and, therefore, proteomics, the study of the proteome, is currently, and will likely remain, a significant experimental approach for mechanistic research in neurotoxicology. The purpose of this review is to discuss proteomics as a tool for neurotoxicological investigations. A variety of classic proteomic techniques (e.g. liquid chromatography (LC)/tandem mass spectroscopy, two-dimensional gel image analysis) as well as more recently developed approaches (e.g. two-hybrid systems, antibody arrays, protein chips, isotope-coded affinity tags, ICAT) are available to determine protein levels, identify components of multiprotein complexes and to detect post-translational changes. Proteomics, therefore, offers a comprehensive overview of cell proteins, and in the case of neurotoxicant exposure, can provide quantitative data regarding changes in corresponding expression levels and/or post-translational modifications that might be associated with neuron injury.

Animals↗

PubMed2024

Secretome Analysis Using Affinity Proteomics and Immunoassays: A Focus on Tumor Biology.

The study of the cellular secretome using proteomic techniques continues to capture the attention of the research community across a broad range of topics in biomedical research. Due to their untargeted nature, independence from the model system used, historically superior depth of analysis, as well as comparative affordability, mass spectrometry-based approaches traditionally dominate such analyses. More recently, however, affinity-based proteomic assays have massively gained in analytical depth, which together with their high sensitivity, dynamic range coverage as well as high throughput capabilities render them exquisitely suited to secretome analysis. In this review, we revisit the analytical challenges implied by secretomics and provide an overview of affinity-based proteomic platforms currently available for such analyses, using the study of the tumor secretome as an example for basic and translational research.

Humans↗

PubMed2006

2-DE proteomic profiling of neuronal stem cells.

Proteomics has become a powerful tool in neuroscience studies. Although numerous human neural stem cells are available for research purposes since many years, there exists only limited information on proteomic data from stable neural stem cell lines. Profiling and functional proteome studies of neuronal stem cells will help to describe the protein inventory as well as protein activity and interactions, subcellular localization and posttranslational modifications. The proteomic analysis of neuronal differentiation processes will elucidate the complex events leading to the generation of different phenotypes via distinctive developmental programs that control self-renewal, differentiation, and plasticity. Using the ReNcell VM197 model, a cell line derived from human fetal ventral mesencephalon stem cells, we studied the protein inventory of the stem cells by 2-DE gel electrophoresis and mass spectrometric protein identification and constructed a 2-DE protein map consisting of more than 400 identified protein spots. This proteome reference database constitutes the basis for further investigations of differential protein expression during differentiation. A profiling of the neuronal differentiation-associated changes displayed the large rearrangement of the proteome during this process, and the proteomic techniques proved to be a valuable tool for the elucidation of neuronal differentiation process and for target protein screening.

Animals↗

PubMed2004

Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium.

Prostate cancer is unusual among neoplasms in that it may be diagnosed at a curable stage through detection of a protein in serum, the serine protease prostate-specific antigen (PSA). PSA is secreted by both normal and neoplastic prostate epithelial cells in response to androgenic hormones and has found widespread use in cancer screening. Because PSA screening is controversial due to sensitivity and specificity issues, efforts continue to focus on the identification and characterization of additional markers that may be used for diagnostic and therapeutic purposes. In this study, we report the application of quantitative proteomic techniques that incorporate isotope coded affinity tag reagents and tandem mass spectrometry to comprehensively identify secreted and cell surface proteins from neoplastic prostate epithelium. LNCaP cells, a prostate tumor-derived cell line that secretes PSA in response to androgen exposure, were grown in a low protein-defined media under androgen-stimulated (A+) and -starved (A-) conditions. Proteomic analysis of the media identified in excess of 600 proteins, 524 of which could be quantified. Nine percent of the proteins had A+/A- ratios > 2.0, including PSA, and 2.5% had ratios < 0.5. A subset of these androgen-regulated proteins appeared to be expressed in abundance. Of these, selected mass spectrometry observations were confirmed by Western analysis. The findings suggest that androgen-mediated release of proteins may occur through the activation of proteolytic enzymes rather than exclusively through transcriptional or translational control mechanisms. On the basis of their known functional roles, several of the abundant androgen-regulated proteins may participate in the progression of neoplastic epithelial cell growth and should be considered as potential serum markers of neoplastic prostate diseases.

Biomarkers, Tumor↗

PubMed2006

Proteomics in diagnostic neuropathology.

In the "postgenome" era, attention has turned to the proteome as a source of complementary diagnostic and prognostic information. Recent advances in imaging mass spectrometry (IMS) uses matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) to acquire up to 1,000 individual protein signals within the molecular weight range of 2,000 to over 100,000 in specific areas of tissue sections. The systematic investigation of these sections permits creation of specific molecular weight images (ion density maps) for each signal detected. Analysis of these images can reveal a collection of unique protein changes, or a "protein signature", of diagnostic and prognostic value. These signatures may also afford assessment of disease progression and tissue response to treatments. Combined with histology and molecular genetic analyses, new proteomic techniques should refine subclassifications and provide defining information about the pathogenesis of many central and peripheral nervous system diseases.

Animals↗

PubMed2004

Proteomics of the Drosophila immune response.

Completion of the Drosophila genome has enabled the use of proteomic approaches for studying complex processes such as the innate immune defense against microorganisms. Microbial infection leads to the activation of responses involving changes at translational and post-translational levels. Proteomics is a tool for assessing such changes in protein expression, localization and post-translational modification. Recently, several studies have reported whole-genome analyses of the Drosophila immune response, both at the transcriptome and proteome levels, leading to a more comprehensive view of fly immunity. In this review, we describe and compare the proteomic techniques used in these analyses and discuss the results obtained by differential protein profiling of the Drosophila immune response.

Animals↗

PubMed2001

Media containing aromatic compounds induce peculiar proteins in Acinetobacter radioresistens, as revealed by proteome analysis.

An Acinetobacter radioresistens strain able to grow on phenol or benzoate as sole carbon and energy source through the beta-ketoadipate pathway was isolated in our laboratories. In previous research, we found a different expression of catechol-1,2-dioxygenase isoenzymes (C-1,2-O) depending on the growth substrate (phenol or benzoate). In the present study, we used proteome techniques to extend our investigation to other enzymes involved in the aromatic degradation pathway. Since the first nontoxic metabolite in this route is cis,cis-muconic acid, we focused our attention on the enzymes leading to this compound, chiefly phenol hydroxylase (PH), benzoate dioxygenase (BD), cis-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate dehydrogenase (D) and C-1,2-O. In particular, the A. radioresistens proteome was monitored under different growth substrate conditions, using acetate, benzoate, or phenol as sole carbon source. We compared the protein maps by software image analysis and detected marked differences, suggesting the inducibility of most enzymes. This research also sought to evaluate the conditions allowing the best expression of enzymes to be used in immobilized systems suitable for bioremediation. The experimental data indicate that benzoate is the best carbon source to gain the highest amount of C-1,2-O and D, while phenol is the best growth substrate to obtain PH.

Acinetobacter↗

PubMed2003

Global analysis of protein expression in yeast.

The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.

Blotting, Western↗

PubMed2001

Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electrophoresis approach prior to mass spectrometry.

A preparative proteomic approach, involving liquid phase isoelectric focusing (IEF) in combination with one-dimensional electrophoresis and electroelution followed by mass spectrometry and database searches, was found to be an important tool for identifying low-abundant proteins (microgram/L) in human cerebrospinal fluid (CSF) and membrane proteins in human frontal cortex. Several neuron-related proteins, such as amyloid precursor-like protein, chromogranins A and B, glial fibrillary acid protein, beta-trace, transthyretin, ubiquitin, and cystatin C, were identified in CSF. Several types of proteins were also characterized from a detergent-solubilized human frontal cortex homogenate including membrane proteins such as synaptophysin, syntaxin and Na+/K+ ATPase. One-third of the identified proteins have not previously been identified in human CSF or human frontal cortex using proteomic techniques. The absence of these proteins in two-dimensional electrophoresis maps might be due to insufficient amounts or low solubility. The advantages of using preparative liquid phase electrophoretic separations for identifying proteins from complex biological mixtures are speed of analysis, high loadability in the IEF separation, nondiscrimination of membrane proteins or low abundance proteins, yielding sufficient amounts for characterization by mass spectrometry. The use of this strategy in proteome studies of CSF/brain tissue is expected to offer new perspectives in studies of the pathology of neurodegenerative diseases, and reveal new potential markers for brain disorders.

Brain Chemistry↗

PubMed2004

The Schistosoma mansoni soluble proteome: a comparison across four life-cycle stages.

Differential analysis of immune responses to schistosomes has routinely been performed using complex mixtures of soluble proteins from various life-cycle stages, on the assumption that these differed significantly in composition. Proteomic techniques now allow us to characterise and compare such mixtures. The soluble proteins from cercariae, lung-schistosomula, adult worms and eggs of Schistosoma mansoni were separated by high-resolution two-dimensional electrophoresis and the resulting images analysed using appropriate software. A high degree of quantitative and qualitative similarity in spot pattern was revealed across the life-cycle, greatest between adjacent stages. To initiate mapping of these soluble proteomes, the 40 most abundant spots in each preparation, accounting for 21-46% of the total protein, were subjected to peptide fingerprinting by mass spectrometry. On average 55% of the spots were identified, but overall, these comprised only 32 different protein species. With one exception all proteins originated in the cytosol and 24 of the 32 had previously been pinpointed by virtue of their immunoreactivity, including four of the WHO priority vaccine candidates. The similarity in composition between the four preparations means that they are unlikely to discriminate adequately between immune responses to different life-cycle stages and argues strongly for the need to identify true stage-specific marker proteins. Equally, it is difficult to reconcile the abundance and immunogenicity of such cytosolic proteins with their status as vaccine candidates, as it is unlikely they will be accessible to the immune system in an intact parasite.

Animals↗