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PubMed2006

Automated quantification of quantum-dot-labelled epidermal growth factor receptor internalization via multiscale image segmentation.

The ability to monitor epidermal growth factor receptor (EGFr) internalization specifically, and cellular protein concentrations and activation states in general, has been recently improved by the use of appropriately functionalized quantum dots (QDs), as a result of the long-lasting fluorescence, brightness and multicolour of these nanoparticles. However, important quantitative information about locational proteomics is based on the analysis of the properties of many cells and cell cultures on a per-cell basis, rather than tracking individual events within one cell. Moreover, relative positional information is often gained from traditional staining protocols of distinct cellular compartments that are prone to noise, fading and low contrast. We apply a novel multiscale image segmentation based on region growing to classify automatically objects in fixed cell preparations and to define regional zones in all cells prior to QD concentration measures. This allows rapid quantitative description of EGFr internalization as it changes with incubation time. The capabilities realizable by simultaneous application of confocal imaging and functionalized QDs in conjunction with advanced image analysis are a prerequisite for automated and multiplexed cytomics assays.

Biotin↗
PubMed2026

Targeted Modulation of Abundant Proteins Enhances Proteomic Profiling of Ovarian Cancer Ascites: A Pilot Technical Workflow Comparison.

Ascites from ovarian cancer patients are increasingly recognized as a valuable biofluid for cancer research, as its protein composition reflects the disease state and may reveal biomarkers of treatment sensitivity and response. However, the detection of low-abundance proteins is hindered by the presence of highly abundant proteins such as albumin. In this study, we evaluated five protein preparation methods for their effectiveness in depleting high-abundance or enriching low-abundance proteins in ovarian cancer ascites. The Norgen (Nor), Minutes (Min), and Perchloric acid (PerCA) methods were based on abundant protein depletion, while the Urine (Uri) and Nanomics (Nano) kits focused on low-abundance protein enrichment. Processed samples were analyzed using label-free quantitative bottom-up proteomics by LC-MS/MS, followed by a bioinformatics assessment. Compared with undepleted ascites (UnD), Min, Nor, Nano, and PerCA increased protein identifications, whereas Uri produced profiles similar to those of UnD. Notably, PerCA and Nano enabled the identification of distinct protein subsets associated with cancer-related pathways, including immune responses and autophagy. PerCA enriched transmembrane and secreted immunomodulatory glycoproteins, whereas Nano enrichment primarily captured secreted, nuclear, and cytoplasmic soluble proteins. Overall, our results show that both high-abundance protein depletion and low-abundance enrichment improve ascites proteome coverage, each offering distinct advantages in identifying biologically relevant low-abundance proteins.

Female↗
PubMed2002

Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli.

Separation and relative quantitation of complex protein mixtures remain two of the most challenging aspects of proteomics. Here an advanced technique called fluorescence difference 2-D gel electrophoresis technology (2D-DIGE) has been applied to a model system study of the Escherichia coli proteome after benzoic acid treatment. The molecular weight and charge matched cyanine dyes enable pre-electrophoretic labelling of control and treated samples which are then mixed and run in the same gel. Pooled control and treated samples labelled with Cy trade mark 3 were used as an internal standard for both Cy5 labelled control and treated E. coli samples. Together with DeCyder trade mark imaging analysis software, more accurate quantitative analysis than conventional two-dimensional polyacrylamide gel electrophoresis was achieved. Using matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight mass spectrometry a total of 179 differentially expressed protein spots were identified. These included enzymes, stress related and substrate (e.g. amino acids, maltose, ribose and TRP repressor) binding proteins. Of the spots analysed, 77% contained only one protein species per spot, hence the change in protein expression measured was solely attributed to the identified protein. Many membrane proteins and protein isoforms were identified indicating both adequate solubilization of E. coli samples and potential post-translational modification. The results indicate that the regulatory mechanisms following benzoic acid treatment of E. coli are far more complicated than hitherto expected.

Benzoic Acid↗
PubMed2005

Characterization of the human blood plasma proteome.

We describe methods for broad characterization of the human plasma proteome. The combination of stepwise immunoglobulin G (IgG) and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of > 94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (< 30 pg/mL to approximately 30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods. More than 80% of the observed proteins demonstrate interactions with IgG and/or albumin, and the human plasma protein loss in the affinity chromatography/strong cation exchange/reversed-phase liquid chromatography-tandem mass spectrometry methodology was investigated in detail. The results of this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies of the identification of novel protein disease markers, as well as further studies of protein-protein interactions.

Animals↗
PubMed2005

Mass spectrometry-based expression profiling of clinical prostate cancer.

The maturation of MS technologies has provided a rich opportunity to interrogate protein expression patterns in normal and disease states by applying expression protein profiling methods. Major goals of this research strategy include the identification of protein biomarkers that demarcate normal and disease populations, and the identification of therapeutic biomarkers for the treatment of diseases such as cancer (Celis, J. E., and Gromov, P. (2003) Proteomics in translational cancer research: Toward an integrated approach. Cancer Cell 3, 9-151). Prostate cancer is one disease that would greatly benefit from implementing MS-based expression profiling methods because of the need to stratify the disease based on molecular markers. In this review, we will summarize the current MS-based methods to identify and validate biomarkers in human prostate cancer. Lastly, we propose a reverse proteomic approach implementing a quantitative MS research strategy to identify and quantify biomarkers implicated in prostate cancer development. With this approach, the absolute levels of prostate cancer biomarkers will be identified and quantified in normal and diseased samples by measuring the levels of native peptide biomarkers in relation to a chemically identical but isotopically labeled reference peptide. Ultimately, a centralized prostate cancer peptide biomarker expression database could function as a repository for the identification, quantification, and validation of protein biomarker(s) during prostate cancer progression in men.

Biomarkers, Tumor↗
PubMed2004

Changes of chondrocyte metabolism in vitro: an approach by proteomic analysis.

Changes in chondrocyte metabolism in vitro using different support systems and under different culture conditions were studied with a proteomic approach. Qualitative and quantitative modifications in the synthesis of chondrocyte proteins were investigated using two-dimensional (2D) gel electrophoresis. This technique provided a simple way to visualize the most abundant chondrocyte proteins. Proteins were identified after in-gel proteolysis with trypsin and matrix-assisted laser desorption ionization-time of flight mass spectrometry, using peptide mass fingerprinting. Tryptic peptide masses were measured and matched against a computer-generated list from the simulated trypsin proteolysis of a protein database (SwissProt).

Animals↗
PubMed2005

Toward the complete membrane proteome: high coverage of integral membrane proteins through transmembrane peptide detection.

To attain a comprehensive membrane proteome of two strains of Corynebacterium glutamicum (l-lysine-producing and the characterized model strains), both sample pretreatment and analysis methods were optimized. Isolated bacterial membranes were digested with trypsin/cyanogen bromide or trypsin/chymotrypsin, and a complementary protein set was identified using the multidimensional protein identification technology (MudPIT). Besides a distinct number of cytosolic or membrane-associated proteins, the combined data analysis from both digests yielded 326 integral membrane proteins ( approximately 50% of all predicted) covering membrane proteins both with small and large numbers of transmembrane helices. Also membrane proteins with a high GRAVY score were identified, and basic and acidic membrane proteins were evenly represented. A significant increase in hydrophobic peptides with distinctly higher sequence coverage of transmembrane regions was achieved by trypsin/chymotrypsin digestion in an organic solvent. The percentage of identified membrane proteins increased with protein size, yielding 80% of all membrane proteins above 60 kDa. Most prominently, almost all constituents of the respiratory chain and a high number of ATP-binding cassette transport systems were identified. This newly developed protocol is suitable for the quantitative comparison of membrane proteomes and will be especially useful for applications such as monitoring protein expression under different growth and fermentation conditions in bacteria such as C. glutamicum. Moreover with more than 50% coverage of all predicted membrane proteins (including the non-expressed species) this improved method has the potential for a close-to-complete coverage of membrane proteomes in general.

Bacterial Proteins↗
PubMed2006

Proteomics approaches to elucidate oncogenic tyrosine kinase signaling in myeloid malignancies.

Myeloid malignancies frequently harbor specific mutations in protein tyrosine kinases leading to oncogenic cell signaling. The most extensively investigated example is chronic myeloid leukemia, where the pathogenic tyrosine kinase fusion protein Bcr-Abl is a successful target for disease control by the specific inhibitor imatinib mesylate. In acute myeloid leukemia the receptor tyrosine kinase Flt3 is frequently mutated and inhibitors to impair the oncogenic signaling are in development. In this review we exemplify oncogenic signaling and how signal pathways can be unraveled with help from proteomics-based technologies. The distinction between cell extract and single cell approaches aiming at rigorous standardization and reliable quantitative aspects for future proteomics-based diagnostics is discussed.

Benzamides↗
PubMed2006

Mitochondrial proteomic characterization of human normal articular chondrocytes.

OBJECTIVE: Mitochondrial dysfunctions have been associated with apoptosis, aging and osteoarthritis (OA). Chondrocyte mitochondrial proteins are attractive targets for the study of the metabolism of cartilage degradation. The copurification of "contaminating" proteins has been the major problem in all phases of mitochondrial proteome research. Therefore, we set up a procedure for the proteomic analysis of human chondrocyte mitochondrial proteins. METHOD: Four types of protein extracts were obtained from primary cultured chondrocytes isolated from healthy donors: (1) initial total chondrocyte extract (CE), (2) cytosol-enriched supernatant fraction (CY), (3) crude mitochondria fraction (CM), and (4) pure mitochondria fraction (PM). Mitochondria were purified by density gradient ultracentrifugation. Mitochondrial proteins were separated by means of two-dimensional gel electrophoresis (2-DE) and silver stained. Protein spots were then identified by mass spectrometry using MALDI-TOF/TOF technology. RESULTS: The best 2-DE reference map of mitochondrial proteome was constructed employing PM fraction. Thirty-nine percent of the identified proteins were functionally distributed in the mitochondria, 14% in the endoplasmic reticulum and 36% in the cytoplasm. Examining their biological function, 22% are involved in protein targeting, 12% in signaling, 12% in glycolysis, 10% in RNA, DNA or protein synthesis, 10% in oxidative phosphorylation and 4% in redox. The analysis of mitochondrial Mn-superoxide dismutase (SODM) revealed an age-dependent decrease of this protein. CONCLUSION: PM fraction allowed the obtention of a high quality proteomic map for the study of mitochondrial proteins in human articular chondrocytes. This proteomic approach may be also efficient to analyze both quantitative and qualitative modulations of the mitochondrial proteome in human chondrocytes during aging and pathological conditions such as OA.

Adolescent↗
PubMed2006

Quantitative and qualitative comparisons of Cryptosporidium faecal purification procedures for the isolation of oocysts suitable for proteomic analysis.

With the recent publication of the Cryptosporidium genome, investigation of the proteins expressed by Cryptosporidium parvum will provide complementary information on the biology of this complex organism. Proteomic studies on this apicomplexan parasite have been hampered due to the inability to culture or isolate high numbers required for 2D gel analysis. Neonatal calves are a common source of Cryptosporidium oocysts and we report on the development of a sucrose-Percoll purification procedure which produced the high yield and purity (free from faecal and bacterial contaminants) that is required for successful proteomic studies from neonatal calves. We report on the development of quantitative and qualitative flow cytometric methods which were confirmed by epifluorescence microscopy. A comparison of five common purification procedures was carried out to determine the efficiency of the sucrose-Percoll gradient. 2D-PAGE results strongly support the sucrose-Percoll procedure as the most suitable method for applications like proteomics which require the recovery of high numbers of isolated oocysts with minimal faecal and bacterial contaminants.

Animals↗
PubMed2007

Topical delivery of silver nanoparticles promotes wound healing.

Wound healing is a complex process and has been the subject of intense research for a long time. The recent emergence of nanotechnology has provided a new therapeutic modality in silver nanoparticles for use in burn wounds. Nonetheless, the beneficial effects of silver nanoparticles on wound healing remain unknown. We investigated the wound-healing properties of silver nanoparticles in an animal model and found that rapid healing and improved cosmetic appearance occur in a dose-dependent manner. Furthermore, through quantitative PCR, immunohistochemistry, and proteomic studies, we showed that silver nanoparticles exert positive effects through their antimicrobial properties, reduction in wound inflammation, and modulation of fibrogenic cytokines. These results have given insight into the actions of silver and have provided a novel therapeutic direction for wound treatment in clinical practice.

Administration, Topical↗
PubMed1998

Proteome and proteomics: new technologies, new concepts, and new words.

The goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations such as disease or drug treatment. Quantitative analysis of protein expression data obtained by high-throughput methods has led us to define the concept of "regulatory homology" and use it to begin to elucidate the basic structure of gene expression control in vivo. Such investigations lay the groundwork for construction of comprehensive databases of mechanisms (cataloguing possible biological outcomes), the next logical step after the soon to be completed cataloguing of genes and gene products. Mechanism databases provide a roadmap towards effective therapeutic intervention that is more direct than that offered by conventional genomics approaches.

Humans↗
PubMed2002

Initial sequencing and comparative analysis of the mouse genome.

The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.

Animals↗
PubMed2006

Discovery of regulatory molecular events and biomarkers using 2D capillary chromatography and mass spectrometry.

An important component of proteomic research is the high-throughput discovery of novel proteins and protein-protein interactions that control molecular events that contribute to critical cellular functions and human disease. The interactions of proteins are essential for cellular functions. Identifying perturbation of normal cellular protein interactions is vital for understanding the disease process and intervening to control the disease. A second area of proteomics research is the discovery of proteins that will serve as biomarkers for the early detection, diagnosis and drug treatment response for specific diseases. These studies have been referred to as clinical proteomics. To discover biomarkers, proteomics research employs the quantitative comparison of peptide and protein expression in body fluids and tissues from diseased individuals (case) versus normal individuals (control). Methods that couple 2D capillary liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC/MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput and high-sensitivity identification of thousands of individual proteins from complex biological samples. The systematic comparison of experimental conditions and controls allows protein function or disease states to be modeled. This review discusses the different purification and quantification strategies that have been developed and used in combination with 2D-LC/MS/MS and computational analysis to examine regulatory protein networks and clinical samples.

Biomarkers↗
PubMed2004

Forward chemical genetics: library scaffold design.

With the unraveling of the entire human genome, it has become imperative to understand the function of the gene products, proteins. Within the past several years, chemical genetics has gained recognition as a powerful approach to study protein function by using small molecules as gene knock-out or knock-in mimics. Forward chemical genetics is a three-step process; the design and synthesis of a small molecule library represents the first step followed secondly by the search for novel phenotypes and then by isolation and identification of target protein(s). This review will focus on the first step, the design of the scaffold for small molecule libraries. It will also examine the connection between the choice of a scaffold and the propensity of that library to demonstrate enhanced biological activity when tested in certain cellular systems.

Animals↗
PubMed2006

[Adeno-associated viral gene transfer of SERCA2a improves heart function in chronic congestive heart failure rats].

OBJECTIVE: To study the therapy effect of adeno-associated viral gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) on chronic congestive heart failure (HF) in 30 days, and the possible mechanism of the therapy effect. METHODS: The rats were divided into four groups: control group, HF group, Group HF + EGFP, and Group HF + SERCA2a. HF rats were obtained by creating descending aortic constriction. 0.9% sodium chloride solution, recombinant adeno-associated virus carrying enhanced green fluorescent protein gene (rAAV2.eGFP) and recombinant adeno-associated virus carrying SERCA2a gene (rAAV2.SERCA2a), were respectively delivered to pericardium of HF rats in different groups by intrapericardial injection with a trans-diaphragmatic approach. 30 days after gene transfer, hemodynamic parameters, SERCA2a protein expression and SERCA2a activity were analyzed. The proteome difference from rat hearts between Groups HF + SERCA2a and HF was detected by expression proteomics. Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms of hearts in different groups were performed at 30 days. RESULTS: At 30 days, left ventricular function improved significantly in HF rats infected with rAAV2.SERCA2a (LVSP 146.52 +/- 13.86 vs 97.91 +/- 12.13, LVEDP 7.88 +/- 2.88 vs 21.15 +/- 3.57, LV +dp/dt 11 206.16 +/- 1730.11 vs 5948.93 +/- 1283.43, LV -dp/dt -8249.54 +/- 1076.09 vs -4497.50 +/- 652.12; P < 0.05). The recovered cardiac function in Group HF + SERCA2a rats was comparable to control rats, and had lower LV-weight/Body-weight ratio (2.46 +/- 0.17 vs 2.71 +/- 0.24, P < 0.05). Overexpression of SERCA2a increased both the protein content (0.39 +/- 0.11 vs 1.11 +/- 0.18, P < 0.05) and activity (228.62 +/- 25.11 vs 82.55 +/- 14.13, P < 0.05) up to nonfailing levels. Expressions of some energy metabolic enzymes in hearts of Group HF + SERCA2a were much higher than those of HF group. They included creatine kinase-muscle, enolase beta, fructose-bisphosphate aldolase, mitochondrial H(+)-ATP synthase alpha subunit, electron transfer flavoprotein alpha-subunit, H(+)-transporting ATP synthase and heart fatty acid binding protein. Downregulation of alpha-MHC and upregulation of beta-MHC in failing hearts were observed. Gene transfer of SERCA2a could increase the expression of alpha-MHC [(74.48 +/- 3.74)% vs (53.57 +/- 2.30)%, P < 0.05], and decrease the expression of beta-MHC [(25.52 +/- 3.74)% vs (46.43 +/- 2.30)%, P < 0.05] in HF rats. The expression profiles of alpha-MHC and beta-MHC and the ratio of alpha-MHC/beta-MHC were similar to those in controls. CONCLUSIONS: Adeno-associated viral gene transfer of SERCA2a can enhance SERCA2a functions, maintain calcium homeostasis, improve cardiac energy metabolism, and normalize the expression of cardiac myosin heavy chain isoforms in HF rats. As a result, the ventricular systolic and diastolic functions can be improved significantly, and the hypertrophy of the heart may be reduced in clinic. Adeno-associated viral gene transfer of SERCA2a demonstrated good therapy effects on HF rats.

Adenoviridae↗
PubMed2005

Quantitative analysis of severe acute respiratory syndrome (SARS)-associated coronavirus-infected cells using proteomic approaches: implications for cellular responses to virus infection.

We present the first proteomic analysis on the cellular response to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. The differential proteomes of Vero E6 cells with and without infection of the SARS-CoV were resolved and quantitated with two-dimensional differential gel electrophoresis followed by ESI-MS/MS identification. Moreover isotope-coded affinity tag technology coupled with two-dimensional LC-MS/MS were also applied to the differential proteins of infected cells. By combining these two complementary strategies, 355 unique proteins were identified and quantitated with 186 of them differentially expressed (at least 1.5-fold quantitative alteration) between infected and uninfected Vero E6 cells. The implication for cellular responses to virus infection was analyzed in depth according to the proteomic results. Thus, the present work provides large scale protein-related information to investigate the mechanism of SARS-CoV infection and pathogenesis.

Amino Acid Sequence↗
PubMed2002

The application of 2D gel-based proteomics methods to the study of breast cancer.

The protein complement of breast cells consists of many thousands of proteins. Recent developments in 2D gel electrophoresis technology have made studies requiring the quantitative analysis of a differential proteome, such as comparison between normal and malignant cells or investigation of drug effects on cells, truly feasible. Computer software plays a central part in the comparisons between multiple gels required for such experiments. In addition, software tools allow patterns of coexpression of proteins to be studied, offering potential insights into protein regulation, interactions, and functions, especially when combined with complementary data on gene expression. In this paper, the technology and limitations of 2D gel-based proteomics are reviewed. Techniques for comparing sets of gels at a global level as well as identifying specific protein features that differentiate gels are discussed. Our own experience of studying the breast cell proteome is used to illustrate the difficulties and achievements of differential proteomics.

Breast Neoplasms↗