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At least 379 records · Page 21Linked to original sources
PubMed2004

Defining the plant disulfide proteome.

There is considerable interest in redox regulation and new targets for thioredoxin and glutaredoxin are now being identified. It would be of great benefit to the field to have a list of all possible candidates for redox regulation--that is all disulfide proteins in plant. We developed a simple and very powerful method for identifying proteins with disulfide bonds in vivo. In this method, free thiols in proteins are fully blocked by alkylation, following which disulfide cysteines are converted to sulfhydryl groups by reduction. Finally, proteins with sulfhydryls are isolated by thiol affinity chromatography. Our method is unique in that membrane proteins as well as water-soluble proteins are examined for their disulfide nature. By applying this method to Arabidopsis thaliana we identified 65 putative disulfide proteins, including 20 that had not previously been demonstrated to be regulated by redox state. The newly identified, possibly redox-regulated proteins include: violaxanthin de-epoxidase, two oxygen-evolving enhancer proteins, carbonic anhydrase, photosystem I reaction center subunit N, photosystem I subunit III, S-adenosyl-L-methionine carboxyl methyltransferase, guanylate kinase, and bacterial mutT homolog. Possible functions of disulfide bonding in these proteins are discussed.

Arabidopsis↗
PubMed2002

Affinity-capture reagents for protein arrays.

The simultaneous identification and quantitative measurement of the production levels of thousands of different proteins in a biological specimen remains an unachieved goal of modern proteomic research. Advances in the development of microarray-based platforms for highly parallel detection of proteins have therefore received a considerable impulse during the last few years. Here, we review the existing reagents for affinity capture of protein targets, as well as the techniques used for their immobilization on solid supports and methods for the detection of binding events, underlining the problems and the opportunities in this continuously evolving research field.

Biotechnology↗
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↗
PubMed2026

Selenoprotein S associates with complexes governing membrane protein biogenesis and translation-associated processes.

Human selenoprotein S (selenos) is part of the integrated cellular stress response and linked to protein quality control and signaling pathways. Consequently, genetic polymorphisms of selenos are associated with increased risks for diabetes, dyslipidemia, and cardiovascular diseases. Determining the specific roles of selenos in these cellular pathways and diseases has been challenging, as selenos associates with a wide range of protein complexes. Thus, to map the cellular functions of selenos and uncover their interconnections, we used affinity purification and in vivo crosslinking to stabilize transient protein interactions, followed by proteomics to record the resulting selenos interactome. Through mapping of selenos protein partners, we found evidence that selenos associates with complexes responsible for the insertion of membrane proteins into the endoplasmic reticulum (ER) bilayer and their connected quality control components. Furthermore, selenos is also part of metabolic, trafficking, and mitochondrial pathways. Notably, proteins involved in translation preferentially associate with selenos when its C-terminal intrinsically disordered segment containing the redox-active motif is accessible. Together, these results identify the C-terminal redox loop of selenos as a central interaction hub connecting translation with ER membrane protein biogenesis and quality control.

Selenoproteins↗
PubMed2007

Pleckstrin homology (PH) domains and phosphoinositides.

PH (pleckstrin homology) domains represent the 11th most common domain in the human proteome. They are best known for their ability to bind phosphoinositides with high affinity and specificity, although it is now clear that less than 10% of all PH domains share this property. Cases in which PH domains bind specific phosphoinositides with high affinity are restricted to those phosphoinositides that have a pair of adjacent phosphates in their inositol headgroup. Those that do not [PtdIns3P, PtdIns5P and PtdIns(3,5)P2] are instead recognized by distinct classes of domains including FYVE domains, PX (phox homology) domains, PHD (plant homeodomain) fingers and the recently identified PROPPINs (b-propellers that bind polyphosphoinositides). Of the 90% of PH domains that do not bind strongly and specifically to phosphoinositides, few are well understood. One group of PH domains appears to bind both phosphoinositides (with little specificity) and Arf (ADP-ribosylation factor) family small G-proteins, and are targeted to the Golgi apparatus where both phosphoinositides and the relevant Arfs are both present. Here, the PH domains may function as coincidence detectors. A central challenge in understanding the majority of PH domains is to establish whether the very low affinity phosphoinositide binding reported in many cases has any functional relevance. For PH domains from dynamin and from Dbl family proteins, this weak binding does appear to be functionally important, although its precise mechanistic role is unclear. In many other cases, it is quite likely that alternative binding partners are more relevant, and that the observed PH domain homology represents conservation of structural fold rather than function.

Binding Sites↗
PubMed2000

The integration of SPR biosensors with mass spectrometry: possible applications for proteome analysis.

The successful integration of biospecific interaction analysis based on surface plasmon resonance and mass spectrometry produces a powerful technique that couples the benefits of sensitive affinity capture and characterization of binding events with the ability to characterize interacting molecules. A variety of biosensors has been used to capture proteins and peptides biospecifically on sensor surfaces, with subsequent analysis using mass spectrometry. Applying this type of analysis to proteomic studies could lead to ligand and protein-complex identification, and might provide clues leading to the identification of pathways.

Amino Acid Sequence↗
PubMed2006

Effects of subminimum inhibitory concentrations of antibiotics on the Pasteurella multocida proteome.

Subminimum inhibitory concentrations (sub-MICs) of antibiotics can be therapeutically effective, but the underlying molecular mechanisms are not well-characterized. We analyzed the Pasteurella multocida proteome response to sub-MICs of amoxicillin, chlortetracycline, and enrofloxacin using isotope-coded affinity tags (ICAT). There were parallel effects on inhibition of growth kinetics and suppression of protein expression by clusters of orthologous groups (COG) categories. Potential compensatory mechanisms enabling antibiotic adaptation were identified, including increased RecA expression caused by enrofloxacin.

Amoxicillin↗
PubMed2002

Peptidomic approaches in proteomic research.

Peptides play a central role in many physiological processes. In order to comprehensively analyze all peptides and small proteins of a whole organism or a subsystem (peptidome), technologies other than 2-D gel electrophoresis are required. Although systematic efforts directed at peptides and peptidomes, comparable to the numerous proteomics projects, are largely lacking to date, a number that employ liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of a variety of different peptide sources. Furthermore, distinct peptide classes, such as antimicrobial peptides or peptides related to metabolic diseases such as diabetes/obesity, are once again receiving attention. Here we discuss peptides in terms of their applicability to serve as diagnostic markers (or more generally as biomarkers), as well as therapeutic targets or lead compounds. There are also a number of technological challenges that need to be overcome in the study of potent animal venoms and plant toxins, both of which are generally peptides and which are discussed as potential lead compounds for therapeutic intervention in diseases such as cancer.

Amino Acid Sequence↗
PubMed2004

Isotope-differentiated binding energy shift tags (IDBEST) for improved targeted biomarker discovery and validation.

Mass spectrometry has proved to be an important tool for protein biomarker discovery, identification and characterization. However, global proteomic profiling strategies often fail to identify known low-abundance biomarkers as a result of the limited dynamic range of mass spectrometry (two to three orders of magnitude) compared with the large dynamic range of protein concentrations in biologic fluids (11 to 12 orders of magnitude for serum). In addition, the number of peptides generated in such methods vastly overwhelms the resolution capacity of mass spectrometers, requiring extensive sample clean-up (e.g., affinity tag, retentate chromatography and/or high-performance liquid chromatography) before mass spectrometry analysis. Baiting and affinity pre-enrichment strategies, which overcome the dynamic range and sample complexity issues of global proteomic strategies, are very difficult to couple to mass spectrometry. This is due to the fact that it is nearly impossible to sort target peptides from those of the bait since there will be many cases of isobaric peptides. IDBEST (Target Discovery, Inc.) is a new tagging strategy that enables such pre-enrichment of specific proteins or protein classes as the resulting tagged peptides are distinguishable from those of the bait by a mass defect shift of approximately 0.1 atomic mass units. The special characteristics of these tags allow: resolution of tagged peptides from untagged peptides through incorporation of a mass defect element; high-precision quantitation of up- and downregulation by using stable isotope versions of the same tag; and potential analysis of protein isoforms through more complete peptide coverage from the proteins of interest.

Biomarkers↗
PubMedDate not supplied

An affinity-based method for the purification of fluorescently-labeled biomolecules.

Due to the difficulty of separating mixtures of labeled and unlabeled biomolecules, a general new method for the affinity purification of modified proteins has been developed. A Sepharose-based solid support bearing beta-cyclodextrin groups was used to capture chromophore-modified proteins selectively, while unmodified proteins remained in solution. After isolation of the resin, the modified proteins were released by treating the sample with a competitive cyclodextrin binder, such as adamantane carboxylic acid. This procedure was demonstrated for several dyes displaying a wide range of spectral characteristics and diverse chemical structures. Preliminary studies have shown that this method can also be used to enrich modified peptide fragments present in proteolytic digests. This technique is anticipated to accelerate the development of new protein modification reactions and could provide a useful tool for proteomics applications.

Affinity Labels↗
PubMed2006

Viral proteomics: a promising approach for understanding JC virus tropism.

The human polyomavirus JC virus (JCV) is responsible for the CNS demyelination observed in cases of progressive multifocal leukoencephalopathy. The JCV regulatory region (promoter) is a hypervariable, noncoding, nucleotide sequence positioned between the early and late protein-coding regions in the viral genome. Selective binding of cellular transcription factors to this promoter region participates in the control of viral tropism. Hence, further study of these proteins might provide new insights into JCV tropism and associated pathogenesis. This review gives an overview of viral proteomics - the study of all proteins expressed from the viral gene transcripts, and all the cellular proteins that play a role in JCV tropism. It also describes a new biochemical approach for studying relevant JCV promoter-binding proteins, which is an anchored-JCV transcriptional promoter (ATP) assay. An ATP assay utilizes the product of PCR-amplified JCV promoter sequences coupled with Sepharose beads in order to capture and isolate cellular nuclear proteins with specific promoter-binding affinity for analysis. Proteins that bind to JCV-ATPs can be eluted and subjected to proteomic analysis. Insights from this approach may improve the understanding of viral and cellular parameters that control JCV tropism.

Deoxyribonuclease I↗
PubMed2006

Identification of yeast oxidized proteins: chromatographic top-down approach for identification of carbonylated, fragmented and cross-linked proteins in yeast.

The effects of oxidative stress on the yeast proteome were studied using hydrogen peroxide as the stress agent. Oxidized proteins were isolated by (1) biotinylation of oxidized proteins with biotin hydrazide, (2) affinity selection using monomeric avidin affinity chromatography, and (3) further fractionated by reversed-phase liquid chromatography (RPLC) on a C(8) column. Oxidized protein fractions from RPLC were then trypsin digested and the peptide cleavage fragments identified by tandem mass spectrometry (MS/MS). Slightly over 400 proteins were identified. Sites of carbonyl formation were found in roughly one fourth of these proteins. Oxidation on other amino acids in carbonylated peptides was seen in 32 cases while carbonylation was absent in 96 of the oxidized proteins observed. Although there are large numbers of potential oxidation sites, oxidation seemed to be restricted to a small area in most of the proteins identified. Sometimes multiple amino acids in the same tryptic peptide were oxidized. A second trend was that more than 8% of the proteins identified appeared in more than one of the RPLC fractions. Based on the position of the peptides identified in the primary structure of protein candidates derived from databases it was concluded that this occurred by fragmentation of a parent protein. It is not clear from the data whether the fragmentation process was of enzymatic or oxidative origin. Finally, peptides from two or more proteins occurred together in more than one reversed phase fraction with 2% of the proteins identified. This data was interpreted to mean that this was the result of protein cross-linking.

Amino Acid Sequence↗
PubMed2003

The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: II. Evaluation of tandem mass spectrometry methodologies for large-scale protein analysis, and the application of statistical tools for data analysis and interpretation.

Proteomic approaches to biological research that will prove the most useful and productive require robust, sensitive, and reproducible technologies for both the qualitative and quantitative analysis of complex protein mixtures. Here we applied the isotope-coded affinity tag (ICAT) approach to quantitative protein profiling, in this case proteins that copurified with lipid raft plasma membrane domains isolated from control and stimulated Jurkat human T cells. With the ICAT approach, cysteine residues of the two related protein isolates were covalently labeled with isotopically normal and heavy versions of the same reagent, respectively. Following proteolytic cleavage of combined labeled proteins, peptides were fractionated by multidimensional chromatography and subsequently analyzed via automated tandem mass spectrometry. Individual tandem mass spectrometry spectra were searched against a human sequence database, and a variety of recently developed, publicly available software applications were used to sort, filter, analyze, and compare the results of two repetitions of the same experiment. In particular, robust statistical modeling algorithms were used to assign measures of confidence to both peptide sequences and the proteins from which they were likely derived, identified via the database searches. We show that by applying such statistical tools to the identification of T cell lipid raft-associated proteins, we were able to estimate the accuracy of peptide and protein identifications made. These tools also allow for determination of the false positive rate as a function of user-defined data filtering parameters, thus giving the user significant control over and information about the final output of large-scale proteomic experiments. With the ability to assign probabilities to all identifications, the need for manual verification of results is substantially reduced, thus making the rapid evaluation of large proteomic datasets possible. Finally, by repeating the experiment, information relating to the general reproducibility and validity of this approach to large-scale proteomic analyses was also obtained.

Amino Acid Sequence↗
PubMed2006

The ROP2 family of Toxoplasma gondii rhoptry proteins: proteomic and genomic characterization and molecular modeling.

Four rhoptry proteins (ROP) of Toxoplasma gondii previously identified with mAb have been affinity purified and analyzed by MS; the data obtained allowed the genomic sequences to be assigned to these proteins. As previously suggested for some of them by antibody crossreactivity, these proteins were shown to belong to a family, the prototype of which being ROP2. We describe here the proteins ROP2, 4, 5, and 7. These four proteins correspond to the most abundant products of a gene family that comprises several members which we have identified in genomic and EST libraries. Eight additional sequences were found and we have cloned four of them. All members of the ROP2 family contain a protein-kinase-like domain, but only some of them possess a bona fide kinase catalytic site. Molecular modeling of the kinase domain demonstrates the conservation of residues critical for the stabilization of the protein-kinase fold, especially within a hydrophobic segment described so far as transmembrane and which appears as an helix buried inside the protein. The concomitant synthesis of these ROPs by T. gondii tachyzoites suggests a specific role for each of these proteins, especially in the early interaction with the host cell upon invasion.

Amino Acid Sequence↗
PubMed2006

In vitro display technologies reveal novel biopharmaceutics.

Display technologies are fundamental to the isolation of specific high-affinity binding proteins for diagnostic and therapeutic applications in cancer, neurodegenerative, and infectious diseases as well as autoimmune and inflammatory disorders. Applications extend into the broad field of antibody (Ab) engineering, synthetic enzymes, proteomics, and cell-free protein synthesis. Recently, in vitro display technologies have come to prominence due to the isolation of high-affinity human antibodies by phage display, the development of novel scaffolds for ribosome display, and the discovery of novel protein-protein interactions. In vitro display represents an emerging and innovative technology for the rapid isolation and evolution of high-affinity peptides and proteins. So far, only one clinical drug candidate produced by in vitro display technology has been approved by the FDA for use in humans, but several are in clinical or preclinical testing. This review highlights recent advances in various engineered biopharmaceutical products isolated by in vitro display with a focus on the commercial developments.

Biopharmaceutics↗
PubMed2006

High-throughput identification of IMCD proteins using LC-MS/MS.

The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.

Animals↗
PubMed2005

Subproteomic analysis of metal-interacting proteins in human B cells.

Metal-protein interactions are vitally important in all living organisms. Metalloproteins, including structural proteins and metabolic enzymes, participate in energy transfer and redox reactions or act as metallochaperones in metal trafficking. Among metal-associated diseases, T cell mediated allergy to nickel (Ni) represents the most common form of human contact hypersensitivity. With the aim to elucidate disease-underlying mechanisms such as Ni-specific T cell activation, we initiated a proteomic approach to identify Ni-interacting proteins in human B cells. As antigen presenting cells, B cells are capable of presenting MHC-associated Ni-epitopes to T cells, a prerequisite for hapten-specific T cell activation. Using metal-affinity enrichment, 2-DE and MS, 22 Ni-interacting proteins were identified. In addition to known Ni-binding molecules such as tubulin, actin or cullin-2, we unexpectedly discovered that at least nine of these 22 proteins belong to stress-inducible heat shock proteins or chaperonins. Enrichment was particularly effective for the hetero-oligomeric TRiC/CCT complex, which is involved in MHC class I processing. Blue Native/SDS electrophoresis analysis revealed that Ni-NTA-beads specifically retained the complete protein machinery, including the associated chaperonin substrate tubulin. The apparent Ni-affinity of heat shock proteins suggests a new function of these molecules in human Ni allergy, by linking innate and adaptive immune responses.

B-Lymphocytes↗
PubMed2001

The scanning near-field optical microscope as a tool for proteomics.

The identification of the entire genetic code of human DNA is more or less completed. With this knowledge, research in identifying the real information lying in the genes, will begin. This information is contained in the proteins, which are the main biological actors in the cell. For this reason proteins will be targeted in biological investigations in the future. The structure, affinity and reactivity of each identified protein has to be determined, which is a primary goal in the field of proteomics. This will require new and better strategies to identify protein-protein interaction. Our approach, based on the detection and visualization of single proteins by scanning near-field optical microscopy (SNOM), has allowed us to visualize various fixed and fluorochrome-labelled proteins at the nanometer scale. Subsequently SNOM may then be developed to efficiently detect the specific behavior of a certain protein in response to other biomolecules.

Bacterial Proteins↗