SEARCH · PubMed Health

Results for “Proteomics techniques”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources

PubMed2006

Antigen-epitope retrieval to facilitate proteomic analysis of formalin-fixed archival brain tissue.

Formalin is a routine fixative facilitating tissue preservation and histopathology. Proteomic techniques require freshly frozen specimens, which are often difficult to procure, and methods facilitating proteomic analysis of archival formalin-fixed brain tissue are lacking. We employed antigen-epitope-retrieval principles to facilitate proteomic analysis of brain tissue that had been fixed and stored in formalin for 3-7 years. Twenty-micrometer-thick cryopreserved OCT-embedded sections from inferior temporal cortex of human (7 years in formalin) or mouse brain specimens (3 years in formalin) were hematoxylin-/eosin-stained. Approximately 16-64-mm2 areas of the tissue sections were manually scraped off slides, or approximately 2 mm2 of human brain cortex was captured off membrane-coated slides using laser microdissection. Tissue was treated using various pH and temperature conditions prior to trypsin digestion and nano-LC-MS/MS. The largest number of proteins were retrieved by solubilization at pH 9 at 95 degrees C for 1 h; treatments at pH 4 or 6 at 25 or 65 degrees C were generally ineffective. Three-year formalin-fixed murine tissue did not yield more proteins compared to human tissue. Use of formalin-fixed tissue for proteomics is an invaluable tool for medical research. The combination of proteomics and microdissection enables selective enrichment and identification of novel, unique, or abundant proteins that may be important in pathogenesis.

Animals↗

PubMed2004

Changes in magnetization transfer MRI correlate with spreading depression-induced astroglial reactivity and increased protein expression in mice.

OBJECTIVE: Gliosis refers to a range of glial cell transformations that vary according to specific brain pathologic states. Disease, however, is not a prerequisite for gliosis because glial reactivity may also be seen in regions of increased physiologic activity. Our study tests the hypothesis that high-field-strength magnetization transfer MRI is a sensitive technique for detecting transient glial reactivity after experimental spreading depression, a relatively benign perturbation unaccompanied by cell injury. MATERIALS AND METHODS: Unilateral neocortical spreading depression was elicited in mouse cerebral hemispheres and confirmed by transcranial blood flow and extracellular potential measurements. After 3 days, mice were imaged at 4 T using magnetization transfer techniques. Astroglial reactivity was determined immunohistochemically, and protein expression in control and experimental hemispheres was compared using proteomic techniques. RESULTS: Sixteen ([mean +/- SD] +/- 3) spreading depressions (n = 10) were recorded in experimental hemispheres. Spreading depression was never observed in control hemispheres. At 3 days, an 8% decrease (p < 0.05, n = 4) in magnetization transfer signal intensity was measured in experimental hemispheres, which was associated with a 37% increase (p < 0.001, n = 4) in the intensity of glial fibrillary acidic protein staining. Proteomic analysis performed 3 days after the induction of spreading depression showed upregulation of at least 56 proteins, including extracellular and intracellular elements. CONCLUSION: Magnetization transfer at 4.0-T MRI is a sensitive method for detecting glial reactivity and changes in protein expression not associated with cell injury. These results suggest magnetization transfer MRI techniques may have potential for detecting glial reactivity in physiologic processes such as learning and in early disease states.

Animals↗

PubMed2004

Proteomic approaches in endometriosis research.

To date, the quest to develop a noninvasive diagnostic test for endometriosis has mostly concentrated on the levels of cytokines and growth factors that are involved in inflammation, angioneogenesis and tissue remodeling, present in serum, peritoneal fluid, endometrium and endometriotic lesions. As this has not yet translated into the development of such a diagnostic test, proteomic techniques are now being employed to identify proteins that are potential biomarkers for the disease. As proteomics allows the comprehensive analysis of complex fluid and tissue samples with good sensitivity and resolution, it has promise in delivering markers associated with endometriosis. Once identified, the challenge will be in translating these markers into a clinically useful test for endometriosis, as the pathophysiology of this disease is unknown and likely to be complex and multifactorial. Also, with variation between individuals and the influences of steroid hormones during the menstrual cycle, it could be difficult to validate findings relating to a single protein or small groups of proteins differentially expressed in the disease state. Proteomic profiling, using mass spectrometry in combination with sophisticated bioinformatics software to identify protein patterns, may be where a significant clinical diagnostic contribution can be made.

Endometriosis↗

PubMed2006

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum.

Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.

Bacterial Proteins↗

PubMed2006

Robust algorithm for alignment of liquid chromatography-mass spectrometry analyses in an accurate mass and time tag data analysis pipeline.

Liquid chromatography coupled to mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) has become a standard technique for analyzing complex peptide mixtures to determine composition and relative abundance. Several high-throughput proteomics techniques attempt to combine complementary results from multiple LC-MS and LC-MS/MS analyses to provide more comprehensive and accurate results. To effectively collate and use results from these techniques, variations in mass and elution time measurements between related analyses need to be corrected using algorithms designed to align the various types of data: LC-MS/MS versus LC-MS/MS, LC-MS versus LC-MS/MS, and LC-MS versus LC-MS. Described herein are new algorithms referred to collectively as liquid chromatography-based mass spectrometric warping and alignment of retention times of peptides (LCMSWARP), which use a dynamic elution time warping approach similar to traditional algorithms that correct for variations in LC elution times using piecewise linear functions. LCMSWARP is compared to the equivalent approach based upon linear transformation of elution times. LCMSWARP additionally corrects for temporal drift in mass measurement accuracies. We also describe the alignment of LC-MS results and demonstrate their application to the alignment of analyses from different chromatographic systems, showing the suitability of the present approach for more complex transformations.

Algorithms↗

PubMed2006

Proteomics characterization of protein adsorption onto hemodialysis membranes.

Protein-adsorptive properties are a key feature of membranes used for hemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membrane's performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with structural feature of the membrane surfaces. Minidialyzers of identical structural characteristics composed of either cellulose diacetate or ethylenevinyl alcohol materials were employed to develop an ex vivo apparatus to investigate protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by 2-DE coupled to both MALDI-TOF mass spectrometry (MS) mass fingerprinting and fragmentation analysis on a nanoLC-MS/MS hybrid instrument. Membrane surface characterization included evaluation of roughness (atomic force microscopy), elemental chemical composition (X-ray-photoelectron-spectroscopy), and hydrophilicity (pulsed nuclear magnetic resonance). The present study identifies a number of different proteins as common or characteristic of filter material interaction, showing that proteomic techniques are a promising approach for the investigation of proteins surface-adsorbed onto hemodialysis membrane. Proteomic analysis enables the characterization of protein layers of unknown composition.

Adsorption↗

PubMed2004

Discovery of protein biomarkers for renal diseases.

Animal models and human studies have been useful in dissecting the molecular mechanisms of renal disease and finding new disease targets; however, translation of these findings to new clinical therapeutics remains challenging. Difficulties with detecting early disease, measuring drug effectiveness, and the daunting cost of clinical trials hampers the development of new therapeutics for renal diseases. Many existing laboratory tests were discovered because of inspired recognition that a particular protein might prove useful in clinical practice. New unbiased genomic and proteomic techniques identify many constituents present in biologic samples and thus may greatly accelerate biomarker research. This review focuses on the steps needed to develop new biomarkers that are useful in laboratory and clinical investigations, with particular focus on new proteomic screening technologies. New biomarkers will speed the laboratory and clinical development of new treatments for renal diseases through mechanistic insights, diagnoses that are more refined, early detection, and enhanced proof of concept testing.

Biomarkers↗

PubMed2003

Proteomic database of wool components.

The separation, classification and identification of wool fibre proteins has been of interest for many years. The purposes of this review are to summarise past work in this area and to evaluate the application of modern proteomic techniques to the identification and characterisation of wool proteins. The current state of knowledge of the wool proteome will also be presented.

Amino Acid Sequence↗

PubMed2005

Proteomic resources: integrating biomedical information in humans.

Recent improvements in high-throughput proteomic technologies have unleashed the potential for generating vast amounts of data. Managing and sharing proteomic data is not an easy task. In this article, we will discuss some of the high-throughput proteomic techniques that are commonly used today. We will also review the major issues in sharing and dissemination of proteomic data and the recent community initiatives to standardize data formats and ontologies. An overview of the web-based resources and databases for analysis of proteomic data is also provided. Integration of disparate proteomic data sources with genomic and transcriptomic data should make systems biology type of approaches feasible in the near future.

Amino Acid Sequence↗

PubMed2006

Serum proteome to predict virologic response in patients with hepatitis C treated by pegylated interferon plus ribavirin.

BACKGROUND & AIMS: Surface-enhanced laser desorption ionization time-of-flight mass spectrometry is a proteomic technique that enables the global profiling of proteins. We used this approach to monitor the kinetics of serum proteome in patients with chronic hepatitis C virus infection receiving a standard bitherapy regimen to predict treatment response. METHODS: Ninety-six patients with chronic hepatitis C were retrospectively selected. All patients received complete treatment with pegylated interferon in combination with ribavirin. Patients had serum sampling before starting treatment and at the end of treatment. Results were validated in an independent cohort of 51 patients. RESULTS: Comparison of protein profiles in pretreatment and after-treatment serum allowed us to characterize 50 protein peaks, the level of which significantly varied. In the group of patients with sustained virologic response, 37 peaks displayed significant variation during treatment, whereas only one peak differed in nonresponders. A logistic regression analysis allowed us to define an algorithm composed of 2 protein peaks (fibrosis stage and genotype) that correctly predicted, in pretreatment serum, response to treatment in 89% of all patients with an area under the receiver operating characteristic curve of 0.92. In the independent testing group, the same difference in proteome kinetics was observed between sustained responders and nonresponders. The algorithm correctly predicted treatment response in 81% of patients in the testing group. CONCLUSIONS: This study suggests that the kinetics of proteome are significantly different in serum of patients according to treatment response. Serum protein profiling allows prediction of response to antiviral treatment in a significant proportion of patients.

Adult↗

PubMed2003

Direct identification of cytochrome P450 isozymes by matrix-assisted laser desorption/ionization time of flight-based proteomic approach.

The main targets of our investigation were cytochrome P450 isozymes (P450), the key enzymes of the hepatic drug-metabolizing system. Current research approaches to the identification of individual P450 forms include specific P450 inhibitors or substrates, antibody-based identification, and mRNA-based expression profiling. All of these approaches suffer from one common disadvantage-they all are indirect methods. On the other hand, current developments in mass spectrometry provide a direct and reliable approach to protein identification with sensitivity in the femtomole or low picomole range. In this study we have used high-accuracy, matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-based peptide mapping to perform direct identification of distinct P450 isozymes in various rat and rabbit liver microsomes. For the first time, the P450 isozyme composition of clofibrate-induced rat and phenobarbital-induced rabbit liver microsomes was determined by peptide mass fingerprinting (PMF). Application of MALDI TOF-based PMF allows differential identification of such highly homologous P450s as CYP2B1 and CYP2B2. We have found that CYP2A10 previously reported only in rabbit olfactory and respiratory nasal mucosa is present in phenobarbital (PB)-induced rabbit liver microsomes. Two other rabbit P450s, earlier identified only by screening a cDNA library, were found to be present in PB-induced rabbit liver microsomes. In summary, direct identification of P450s by proteomic technique offers advantages over other methods with regard to identification of distinct P450 isozymes and should become a standard approach for characterizing microsomes.

Animals↗

PubMed2006

Proteomic analysis of opsins and thyroid hormone-induced retinal development using isotope-coded affinity tags (ICAT) and mass spectrometry.

PURPOSE: Analyses that reveal the relative abundance of proteins are informative in elucidating mechanisms of retinal development and disease progression. However, popular high-throughput proteomic methods do not reliably detect opsin protein abundance, which serve as markers of photoreceptor differentiation. We utilized thyroid-hormone (TH) treatment of rainbow trout (Oncorhynchus mykiss) as a model of cone apoptosis and cone regeneration. We used this model to investigate if emerging proteomic technology allows effective analysis of retinal development and opsin protein abundance. We also sought to begin a characterization of proteomic changes in the retina occurring with TH treatment and address whether TH affects proliferation or photoreceptor differentiation. METHODS: Retinal homogenates were prepared from control and TH-treated fish. Peptides from control and treated homogenates were differentially labeled, using isotope-code affinity tags (ICAT) and analyzed using capillary liquid chromatography-electrospray ionization-tandem mass spectrometry (capLC-ESI-MS/MS). This method identifies proteins and quantifies their relative abundance between two samples. RESULTS: The relative abundance of many retinal proteins changed during TH treatment. These included proteins from every functional class. We detected 1,684 different peptides, and our quantification suggests that 94 increased and 146 decreased in abundance more than 50% during TH treatment. Cell-cycle proteins appear to be increased, consistent with TH-inducing cell proliferation, similar to its effect in Xenopus. Other proteins associated with retinal development, such as deltaA and tubulins, changed in abundance during TH treatment. Rod opsin and three cone opsins were identified and the relative abundance of each changed with TH treatment. CONCLUSIONS: ICAT and capLC-ESI-MS/MS are an effective complement to other molecular approaches that investigate the mechanisms of retinal development. Unlike other proteomic techniques, this approach does not require development of species- or tissue-specific methodology, such as characterizing two dimensional (2D) gels or antibodies, in order to be practical as a high-throughput approach. Importantly, this technology was able to assess the relative abundance of opsin proteins. These findings represent the first high-throughput proteomic analysis of the retina and demonstrate the technique's ability to provide useful information in retinal development.

Animals↗

PubMed2003

A functional proteomics approach to signal transduction.

The purpose of this review is to highlight how proteomics techniques can be used to answer specific questions related to signal transduction in a wide variety of systems. In our laboratory, we utilize proteomic technologies to elucidate signal transduction pathways involved in smooth muscle contraction and relaxation, cell growth and tumorigenesis, and the pathogenesis of malaria. We see the real application of this technology as a tool to enhance the power of existing approaches such as classical yeast and mouse genetics, tissue culture, protein expression systems, and site-directed mutagenesis. Our basic approach is to examine only those proteins that differ by some variable from the control sample. In this way, the number of proteins to be processed by electrophoresis, Edman degradation, or mass spectrometry is greatly reduced. In addition, since only those proteins that change in response to a given biological treatment are analyzed, the experimental outcome provides information about specific signaling pathways. Examples of typical experiments in our laboratory are measurement of changes in protein phosphorylation in response to treatment of cells with growth factors or specific drugs, characterization of proteins associated with a bait protein in a "pull-down" experiment, or measurement of changes in protein expression. Frequently, in these experiments, it is necessary to define complex protein mixtures. To achieve this goal, we utilize a variety of techniques to isolate specific types of proteins or "subproteomes" for further analysis. In this review, we discuss strategies used in our laboratory for studying signaling pathways, including subproteome isolation, proteome mining, and analysis of the phosphoproteome.

Amino Acid Sequence↗

PubMed2000

Proteomics of the chloroplast: experimentation and prediction.

New technologies, in combination with increasing amounts of plant genome sequence data, have opened up incredible experimental possibilities to identify the total set of chloroplast proteins (the chloroplast proteome) as well as their expression levels and post-translational modifications in a global manner. This is summarized under the term 'proteomics' and typically involves two-dimensional electrophoresis or chromatography, mass spectrometry and bioinformatics. Complemented with nucleotide-based global techniques, proteomics is expected to provide many new insights into chloroplast biogenesis, adaptation and function.

Chloroplasts↗

PubMed2006

Proteomics of breast cancer: principles and potential clinical applications.

Progresses in screening, early diagnosis, prediction of aggressiveness and of therapeutic response or toxicity, and identification of new targets for therapeutic will improve survival of breast cancer. These progresses will likely be accelerated by the new proteomic techniques. In this review, we describe the different techniques currently applied to clinical samples of breast cancer and the most important results obtained with the two most popular proteomic approaches in translational research (tissue microarrays and SELDI-TOF).

Breast Neoplasms↗

PubMed2005

Toward functional proteomics of alveolar macrophages.

Alveolar macrophages (AM) belong to a phenotype of macrophages with distinct biological functions and important pathophysiological roles in lung health and disease. The molecular details determining AM differentiation from blood monocytes and AM roles in lung homeostasis are largely unknown. With the use of different technological platforms, advances in the field of proteomics have made it possible to search for differences in protein expression between AM and their precursor monocytes. Proteome features of each cell type provide new clues into understanding mononuclear phagocyte biology. In-depth analyses using subproteomics and subcellular proteomics offer additional information by providing greater protein resolution and detection sensitivity. With the use of proteomic techniques, large-scale mapping of phosphorylation differences between the cell types have become possible. Furthermore, two-dimensional gel proteomics can detect germline protein variants and evaluate the impact of protein polymorphisms on an individual's susceptibility to disease. Finally, surface-enhanced laser desorption and ionization (SELDI) time-of-flight mass spectrometry offers an alternative method to recognizing differences in protein patterns between AM and monocytes or between AM under different pathological conditions. This review details the current status of this field and outlines future directions in functional proteomic analyses of AM and monocytes. Furthermore, this review presents viewpoints of integrating proteomics with translational topics in lung diseases to define the mechanisms of disease and to uncover new diagnostic and therapeutic targets.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2005

Identification of inducible protein complexes in the phenol degrader Pseudomonas sp. strain phDV1 by blue native gel electrophoresis and mass spectrometry.

Pseudomonas sp. strain phDV1, being a phenol degrading bacterium, has been found to utilize phenol as sole carbon source via the meta pathway. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes involved in the aromatic degradation pathway. In particular, the Pseudomonas sp. strain phDV1 proteome was monitored under two different growth substrate conditions, using glucose or phenol as sole carbon source. The protein complexes map was compared by BN-PAGE after fractionation by sucrose density centrifugation of the cell extracts. Multiple differences were detected. Further, analysis and identification of the subunit composition of these complexes was carried out using MALDI-TOF MS, allowing the identification of 49 proteins. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. Application of this functional proteomics method resulted in an higher number of the identified proteins.

Bacterial Proteins↗

PubMed2004

Identification of porcine oocyte proteins that are associated with somatic cell nuclei after co-incubation.

Relatively little is known with respect to the oocyte proteins that are involved in nuclear reprogramming of somatic cells in mammals. The aim of the present study was to use a cell-free incubation system between porcine oocyte proteins and somatic cell nuclei and to identify oocyte proteins that remain associated with these somatic cell nuclei. In two separate experiments, porcine oocytes were either labeled with biotin to label total proteins at the germinal vesicle stage or metaphase II stage or they were labeled with 0.1 mM (35)S-methionine either during the first 6 h or 22-28 h of in vitro maturation to characterize protein synthesis during two distinct phases. To determine which oocyte proteins associate with somatic nuclei, labeled proteins were incubated in a collecting buffer and energy-regenerating system with isolated ovarian epithelial-like cell nuclei. After incubation, the nuclei were subjected to a novel affinity-binding system to recover biotin-labeled oocyte proteins or two-dimensional SDS-PAGE for separation and visualization of radiolabeled proteins. Proteins of interest were sent for identification using either matrix-assisted laser desorption/ionization time of flight or liquid chromatography-tandem mass spectrometry. Of the proteins that remain associated with isolated nuclei after incubation, 4 were identified using the affinity-binding system and 24 were identified using mass spectrometry and the two-dimensional gel interface. This study has identified porcine oocyte proteins that associate with somatic cell nuclei in a cell-free system using proteomics techniques, providing a novel way to identify oocyte proteins potentially functionally involved in nuclear reprogramming.

Animals↗