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PubMed2005

The peptidomics concept.

Peptides are a paramount example of how nature diversifies from one single gene to release multiple, regulated functionalities at the desired sites and time. To achieve this, peptides are sequentially generated by a complex network of more than 500 proteases, acting at intracellular sites, upon secretion, in extracellular environments, and, finally, serving (regulated) degradation. This cycle of maturation, activation, and degradation points out that the peptidome is mechanistically linked to the proteome: the distribution between both is regulated by proteases and counter-regulated by protease inhibitors. Given the high diversity of peptides in living systems and their involvement in key regulatory processes, a need for improved peptide discovery, ideally combining peptide sequence identification with peptide profiling, has emerged. Standard proteomic approaches are not suitable for a systematic peptide analysis, since they do not cover the low molecular mass window. The new direction in proteomic research to analyse this "terra incognita" is peptidomics. This novel concept aims at the comprehensive visualization and analysis of small polypeptides, thus covering the mass range between proteomics and metabonomics. The pacemakers for the development of peptidomics technologies are modern mass spectrometry and bioinformatics. They are ideally suited for sensitive and comprehensive peptide analysis, especially in combination with the massive information content of today's genomic and transcriptomic databases. Given the high diversity of native peptides in living systems, clinical chemistry and modern medicine are the prime application areas. The discovery of relevant peptide biomarkers and drug targets will strongly benefit from peptidomics.

Animals↗
PubMed2000

Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics.

Novel and powerful technologies such as DNA microarrays and proteomics have made possible the analysis of the expression levels of multiple genes simultaneously both in health and disease. In combination, these technologies promise to revolutionize biology, in particular in the area of molecular medicine as they are expected to reveal gene regulation events involved in disease progression as well as to pinpoint potential targets for drug discovery and diagnostics. Here, we review the current status of these technologies and highlight some studies in which they have been applied in concert to the analysis of biopsy specimens.

Animals↗
PubMed2006

[Proteomic approach to the effect of epirubicin on hepatoma cells at subcellular level].

Epirubicin is an antineoplastic agent known as an anthracycline. It acts directly on DNA by blocking its replication and transcription, so apoptosis can be induced for cancer cells. The protein expression of cancer cells will be altered due to the induction of pharmacological action of epirubicin, so it is important to pay attention to the altered profiling of proteins. Here proteomic strategy was applied to the hepatoma cells at subcellular level, comparative proteome analysis of mitochondria and nucleus were conducted between the hepatoma cells administered with epirubicin and the not-administered. Centrifugation was used for the subcellular fractionation, then 2-DE for the separation of proteins, imaging analysis for the diction of expression-altered spots, and MALDI-TOF-MS for the identification of proteins. In total, 15 proteins were found to have altered their expression after the induction of epirubicin, among them, 5 proteins showed up-regulated expression and 10 showed down-regulated expression. These altered proteins are involved in life processes of cells such as energy metabolism, protein biosynthesis, structure of cell skeleton, processing and maturation of mRNA, heat shock of cells and apoptosis.

Carcinoma, Hepatocellular↗
PubMed2006

Impact of analytical bias in metabonomic studies of human blood serum and plasma.

Concurrent with the explosion in the number of publications reporting biomarker discovery by profiling technologies, such as proteomics and pattern recognition, has been the increase in evidence highlighting the susceptibility of these approaches to analytical and experimental bias. The work presented here addresses these timely issues by delivering a detailed characterization of the effect of common sources of bias in clinical studies on serum and plasma profiles generated by a key technology in metabonomics, NMR spectroscopy. Specifically, differences in composition when blood samples were collected onto and in the absence of ice, over a series of serum-clot contact times, the stability of NMR-prepared samples over time and the effect on the metabolic profile of freeze-thawing were examined. While differences between individuals were far greater than variation from any other experimental factor, each of the conditions examined did cause slight alterations to the NMR profile that could produce a systematic bias. Variation due to clotting time caused changes in energy metabolites, which were delayed by ice with no other spectral effects. Room-temperature stability and hence NMR spectral repeatability were high (<1% intrasample variation). Higher molecular weight species such as lipoproteins were more susceptible to the variations present in the examined factors. These observations have implications for profiling study design, and hence, our results form a new and valuable resource for those attempting clinical metabolic profiling, for regulatory agencies involved in the licensing of clinical tests and in the generation of international reporting standards for metabonomics.

Humans↗
PubMed2006

Better understanding of organ dysfunction requires proteomic involvement.

Organ dysfunction is defined as a systemic consequence of acute and chronic diseases, a critical and important phase of disease development. The mortality of patients with severe illness is highly correlated with the number and duration of dysfunctional organs. There is still not an efficient and specific therapy to improve the prognosis of patients with organ dysfunction, due to the complexity and severity of the disease. There is a great need to understand molecular mechanisms of the disease, identify disease-related biomarkers, and validate therapeutic effects. Thus, it is important to have a special attention from proteomic scientists to explore the combination between advanced proteomic biotechnology, clinical proteomics, tissue imaging and profiling, and organ dysfunction score systems, to improve the clinical outcomes of these patients.

Biomarkers↗
PubMed2004

All about DIGE: quantification technology for differential-display 2D-gel proteomics.

2D polyacrylamide gel electrophoresis has been the traditional workhorse of proteomics, allowing for the resolution of several thousand proteins in a single gel. Difference gel electrophoresis is an emerging technology that allows for accurate quantification with statistical confidence while controlling for nonbiologic variation, and also increases the dynamic range and sensitivity of traditional 2D polyacrylamide gel electrophoresis. With inclusion of an internal standard formed from equal amounts of every sample in an experiment, difference gel electrophoresis technology also allows for repetitive measurements and multivariable analyses to be quantitatively analyzed in one co-ordinated experiment, yielding statistically-significant changes in protein expression related to many disease states. This technique promises to be an important tool in clinical proteomics and the study of the mechanism of disease, investigating diagnostic biomarkers and pinpointing novel therapeutic targets.

Animals↗
PubMed2005

The effect of maternal asthma on placental and cord blood protein profiles.

OBJECTIVE: We conducted a comparative proteomic analysis of placental and umbilical cord blood proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS) to examine the associations among asthma, fetal gender, and protein profiles. METHODS: Placental tissue and umbilical vein plasma were collected from 10 healthy and 20 asthmatic women. Placental proteins were extracted using phosphate-buffered saline containing protease inhibitors. Samples were applied to the surfaces of strong anion exchange (SAX2), weak cation exchange (WCX2) and immobilized metal affinity capture (IMAC-Cu(2+)) chips. Mass analysis was conducted using a Ciphergen Protein Biology System IIc (Freemont, CA), and differences in individual peak intensities between groups were determined. RESULTS: Fourteen placental peaks were significantly different between asthmatic and non-asthmatic women (seven more highly expressed and seven less highly expressed). Ten umbilical cord blood peak differences were identified, with four peaks more highly expressed and six peaks less highly expressed in asthmatics. Four placental and three umbilical cord blood proteins differed significantly between male and female fetuses. Two placental and five umbilical cord blood peaks were specifically increased in a subgroup of samples collected from asthmatic women who did not use inhaled glucocorticoids and were pregnant with a female fetus, a group previously found to have altered placental function. CONCLUSIONS: This study demonstrates the abilities of the SELDI technique as a tool for protein profiling in tissue or plasma. Further work to positively identify the candidate peptides found in this study may provide a greater understanding of the placental mechanisms leading to alterations in fetal growth in patients with bronchial asthma.

Adult↗
PubMed2001

Proteomics of breast cancer for marker discovery and signal pathway profiling.

Breast cancer is the most common form of cancer among women and the identification of markers to discriminate tumorigenic from normal cells, as well as the different stages of this pathology, is of critical importance. Two-dimensional electrophoresis has been used before for studying breast cancer, but the progressive completion of human genomic sequencing and the introduction of mass spectrometry, combined with advanced bioinformatics for protein identification, have considerably increased the possibilities for characterizing new markers and therapeutic targets. Breast cancer proteomics has already identified markers of potential clinical interest (such as the molecular chaperone 14-3-3 sigma) and technological innovations such as large scale and high throughput analysis are now driving the field. Methods in functional proteomics have also been developed to study the intracellular signaling pathways that underlie the development of breast cancer. As illustrated with fibroblast growth factor-2, a mitogen and motogen factor for breast cancer cells, proteomics is a powerful approach to identify signaling proteins and to decipher the complex signaling circuitry involved in tumor growth. Together with genomics, proteomics is well on the way to molecularly characterizing the different types of breast tumor, and thus defining new therapeutic targets for future treatment.

14-3-3 Proteins↗
PubMed2004

Activity-based probes for protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) are involved in the regulation of many aspects of cellular activity including proliferation, differentiation, metabolism, migration, and survival. Given the large number and complexity of PTPs in cell signaling, new strategies are needed for the integrated analysis of PTPs in the whole proteome. Unfortunately, the activities of many PTPs are tightly regulated by posttranslational mechanisms, limiting the utility of standard genomics and proteomics methods for functional characterization of these enzymes. To facilitate the global analysis of PTPs, we designed and synthesized two activity-based probes that consist of alpha-bromobenzylphosphonate as a PTP-specific trapping device and a linker that connects the trapping device with a biotin tag for visualization and purification. We showed that these probes are active site-directed irreversible inactivators of PTPs and form a covalent adduct with PTPs involving the active site Cys residue. Additionally, we demonstrated that the probes are extremely specific toward PTPs while remaining inert to other proteins, including the whole proteome from Escherichia coli. Consequently, these activity-based PTP probes can be used to profile PTP activity in complex proteomes. The ability to interrogate the entire PTP family on the basis of changes in their activity should greatly accelerate both the assignment of PTP function and the identification of potential therapeutic targets.

Binding Sites↗
PubMed2002

[Recent advances in molecular diagnostic tests].

Recent advances in molecular biotechnologies have facilitated laboratory use of molecular diagnostic tests. Automated systems for amplification and detection, and lately for extraction, of nucleic acid sequences have been developed. The automated systems have allowed improvement of not only assay efficiency but also quality control of the tests. The information on the genome sequence from the human genome project has been studied to elucidate functions of genes and proteins and the clinical significance of nucleic acid sequences with post-genomics such as expression profiling using DNA microarray, proteomics, and single nucleotide polymorphism analysis, in conjunction with bioinformatics. Now the challenge is the development and application of these new technologies as clinical diagnostic tools. Design of a diagnostic array or panel must be developed with defined sequences based on interpretation of a huge quantity of experimental data to meet clinical demands. There is a need for development of generally available instruments that are inexpensive, practical and more importantly reproducible and reliable.

Computational Biology↗
PubMed2005

Progression of head and neck squamous cell cancer.

Squamous cell cancer in the head and neck region (HNSC) is unique concerning its progression since it remains locoregional for long time and visceral metastases develop only in a later stage of the disease. Accordingly, molecular markers of the local invasion and the lymphatic dissemination both have critical importance. HNSC progression is associated with deregulated control of cell proliferation and apoptosis but it seems equally significant the disregulation of the proteolytic machineries. Here we outline the lymphatic metastatic cascade for HNSC to depict key molecular determinants as possible prognostic factors or therapeutic targets identifying immunological selection as a major feature. Unlike in local spreading, invasive potential of cancer cells seems to be less significant during lymphatic dissemination due to the anatomical properties of the lymphatic vessels and tissues. There is a general believe that HNSC is one disease however, data indicate that the anatomical localization of the tumor (the "soil") such as oral, lingual, glottic or pharyngeal has a significant effect on the gene expression profile and corresponding biological behavior of HNSC. Furthermore, even the endocrine milieu of the host was proved to be influential in modulating the progression of HNSC. Gene expression profiling techniques combined with proteomics could help to define and select usefull genetic and biomarkers of progression of HNSC, some of them could well be potential novel therapeutic target.

Animals↗
PubMed2005

Differential protein expression profiles in anterior gills of Eriocheir sinensis during acclimation to cadmium.

Using a proteomic approach, we characterized different protein expression profiles in anterior gills of the Chinese mitten crab, Eriocheir sinensis, after cadmium (Cd) exposure. Two experimental conditions were tested: (i) an acute exposure (i.e. 500 microg Cd l(-1) for 3 days) for which physiological, biochemical and ultrastructural damage have been observed previously; (ii) a chronic exposure (i.e. 50 microg Cd l(-1) for 30 days) resulting in physiological acclimation, i.e. increased resistance to a subsequent acute exposure. Two-dimensional gel electrophoresis (2-DE) revealed six protein spots differentially expressed after acute, and 31 after chronic Cd exposure. From these spots, 15 protein species were identified using MS/MS micro-sequencing and MS BLAST database searches. Alpha tubulin, glutathione S-transferase and crustacean calcium-binding protein 23 were down-regulated after an acute exposure, whereas another glutathione S-transferase isoform was up-regulated. Furthermore, analyses revealed the over-expression of protein disulfide isomerase, thioredoxin peroxidase, glutathione S-transferase, a proteasome subunit and cathepsin D after chronic exposure. Under the same condition, ATP synthase beta, alpha tubulin, arginine kinase, glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase were down-regulated. These results demonstrate that acute and chronic exposure to waterborne Cd induced different responses at the protein expression level. Protein identification supports the idea that Cd mainly exerts its toxicity through oxidative stress induction and sulfhydryl-group binding. As a result, analyses showed the up-regulation of several antioxidant enzymes and chaperonins during acclimation process. The gill proteolytic capacity seems also to be increased. On the other hand, the clearly decreased abundance of several enzymes involved in energy transfer suggests that chronic metal exposure induced an important metabolic reshuffling.

Acclimatization↗
PubMed2004

Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots.

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.

Albumins↗
PubMed2003

Interrogating mouse mammary cancer models: insights from gene expression profiling.

Numerous mouse models for mammary cancer have been developed and characterized based upon their biological, molecular, and histopathological features. In an effort to dissect the molecular anatomy of such models and compare their gene expression profiles to those of human breast cancer, six models representing various oncogenic pathways have been investigated using cDNA microarray technology. Results of these analyses are presented and discussed in the context of technological challenges presented by analyzing data on such a large scale. Further expression profiling coupled with emerging proteomic technologies will more completely define and distinguish mouse models of mammary cancer from each other and provide a comprehensive basis for comparing such models with the human disease they are intended to represent.

Animals↗
PubMed2026

Social disconnection integrates genetic and proteomic risks in suicidal ideation and depression.

Suicidal ideation (SI) and major depressive disorder (MDD) are complex psychiatric conditions arising from the interplay of genetic liability, molecular processes, and psychosocial factors. While these dimensions have been extensively studied in isolation, their joint contribution to SI and MDD remains unclear. This study integrates multi-modal data to elucidate these synergistic effects and develop robust models for individual-level risk stratification. Leveraging longitudinal multi-modal data from 13,085 UK Biobank participants, we integrated genomic, proteomic, and social connection profiles. We developed interpretable risk scores using a rigorous supervised machine learning framework encompassing diverse linear and ensemble classifiers. Permutation importance was employed to quantify feature contributions and derive transparent, weighted risk metrics across diverse classifiers. These scores were validated through association, interaction, and mediation analyses. Social connection-based risk scores significantly differentiated cases and controls across the two suicidal ideation phenotypes at 2017 and 2023 with cross-sectional analyses (AUCs: 0.70 - 0.73), outperforming proteomic-only models. Functional dimensions of social connection emerged as the most informative predictors. Longitudinal analyses revealed that social risk scores at baseline predicted suicidal ideation onset six years later, independent of demographic covariates. Interaction analyses demonstrated that polygenic risk for suicide attempt significantly interacted with both social and proteomic risk features in relation to depression. Structural equation models further confirmed that social disconnection acts as a key mediator linking genetic predisposition to MDD and SI. Social disconnection is a critical risk factor mediating the impact of genetic vulnerability on psychiatric outcomes. Integrating social, genetic, and molecular data supports a multilevel framework for risk stratification and highlights the potential of socially oriented interventions to mitigate biological risk.

Humans↗
PubMed2006

Cell type-specific gene expression profiling in plants by using a combination of laser microdissection and high-throughput technologies.

Laser microdissection (LM) allows for the isolation of specific cells of interest from heterogeneous tissues under direct microscopic visualization with the assistance of a laser beam. By permitting global analyses of gene expression and metabolites in the selected cells, it is a powerful tool for understanding the biological processes in individual cell types during development or in response to various stimuli. Recently, LM technology has been successfully applied to the separation of individual plant cell types. Here, we provide an overview of applications of LM combined with high-throughput technologies including transcript analyses [microarrays, serial analysis of gene expression (SAGE) and 454-sequencing], proteomic analyses and metabolomic profiling, for cell type-specific gene expression analyses in plants.

Gene Expression Profiling↗
PubMed2007

A new analytical material-enhanced laser desorption ionization (MELDI) based approach for the determination of low-mass serum constituents using fullerene derivatives for selective enrichment.

60]fullerene derivatives (dioctadecyl methano[60]fullerene, [60]fullerenoacetic acid, and IDA-[60]fullerene) were prepared and subjected to a comprehensive characterization study including protein binding properties and capacity. These fullerene derivatives were successfully applied as material-enhanced laser desorption/ionization (MELDI) carrier materials. It is shown that diverse functionalities result in characteristic human serum peak patterns (m/z 2000-20 000) in terms of signal intensity as well as the number of detectable masses. In addition, the fullerene derivatives clearly provided differences in the low molecular weight mass region (m/z 1000-4000) after elution of the adsorbed serum constituents, and [60]fullerenoacetic acid was the most effective carrier material. Novel high-speed, monolithic, high-resolution capillary columns, prepared by thermally initiated copolymerization of methylstyrene (MSt) and 1,2-bis(p-vinylphenyl)ethane (BVPE) were employed for eluate separation and target spotting. Thus, serum compounds in the low-mass range were successfully fractionated and subjected to MALDI-MS/MS analysis. This contribution, hence, proposes a new "top-down" strategy for proteome research enabling protein profiling as well as biomarker identification in the low-mass range using selective enrichment, high-resolution separation, and offline MALDI-MS/MS evaluation.

Adsorption↗
PubMed2003

Searching for genes involved in arteriosclerosis: proteomic analysis of cultured human umbilical vein endothelial cells undergoing replicative senescence.

It is known that replicative senescence of endothelium in vivo contributes at least partially to age-related vascular disorders such as arteriosclerosis. However, the genes involved in this process remain to be identified. In this study, we employed a proteomics-based approach to identify candidate genes using in vitro cultured human umbilical vein endothelial cells (HUVECs) as an experimental model for replicative senescence. By comparing protein spots from young and senescent HUVECs using two-dimensional electrophoresis, we identified three up-regulated proteins and five down-regulated proteins in senescent HUVECs as compared to young HUVECs, whose alteration was not observed during replicative senescence of primary human fibroblasts. Consistent results were obtained in Western blotting analysis using specific antibodies raised against some of these proteins, whereas there were no significant changes in the mRNA levels of these genes during senescence of HUVECs. Among them, cathepsin B, a protease participating in both intracellular proteolysis and extracellular matrix remodeling was observed to be dramatically up-regulated in senescent HUVECs and whose activity is known to be up-regulated in atherosclerotic lesions with senescence-associated phenotypes in vivo. Additional proteins, including cytoskeletal proteins and proteins involved in the processes of synthesis, turnover and modification of protein, were identified, whose function in endothelium was previously unsuspected. These proteins identified by a proteomics-based approach using cultured HUVECs may be involved not only in replicative senescence but also in functional alterations in vascular endothelial cells with senescence-associated phenotypes and may serve as molecular markers for these processes.

Arteriosclerosis↗