Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “affinity proteomics”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

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

At least 613 records · Page 34Linked to original sources
PubMed2005

Proteomic tracking of serum protein isoforms as screening biomarkers of ovarian cancer.

Epithelial ovarian cancer is the fourth leading cause of cancer death among women. Due to the asymptomatic nature and poor survival characteristic of the disease, screening for specific biomarkers for ovarian cancer is a major health priority. Differentially expressed proteins in the serum of ovarian cancer patients have the potential to be used as cancer-specific biomarkers. In this study, proteomic methods were used to screen 24 serum samples from women with high-grade ovarian cancer and compared to a control group of 11 healthy women. Affigel-Blue treated serum samples were processed either by linear (pH 4-7) or narrow range (pH 5.5-6.7) IEF strips for the first dimension. Proteins separated in first dimension were resolved by 8-16% gradient SDS-PAGE. Protein spots were visualized by SYPRO Ruby staining, imaged by FX-imager and compared and analyzed by PDQuest software. Twenty-two protein spots were consistently differentially expressed between normal and ovarian cancer patients by resolving proteins in a linear pH strip of 4-7 for the first dimension. Six of the protein spots, significantly up-regulated in grade 3 ovarian cancer patients (p < 0.05), were identified by MALDI-TOF MS and Western blotting as the isoforms of haptoglobin precursor. When serum proteins were resolved on narrow pH range strips (5.5-6.7), 23 spots were consistently differentially expressed between normal and grade 3 ovarian cancer patients. Of these, 4 protein spots significantly down regulated in grade 3 ovarian cancer patients (p < 0.05) were identified by MALDI-TOF MS and Western blotting, as isoforms of transferrin precursor. Increased expression of serum haptoglobin and transferrin was also identified in peritoneal tumor fluid obtained from women diagnosed with grade 2/3 ovarian cancer (n = 7). Changes in the expression of haptoglobin and transferrin in the serum of women with different pathological grades of ovarian cancer was examined by one-dimensional Western blotting method. Serum samples collected from women suffering from benign, borderline, grade 1, grade 2 and grade 3 cancer (n = 4 for haptoglobin and n = 5 for transferrin in each group) were analyzed and compared to the serum of normal healthy women. The mean serum haptoglobin expression in grade 3 ovarian cancer patients was fourfold higher than in the control subjects (p < 0.05). On the other hand, transferrin expression in grade 3 ovarian cancer patients was decreased by twofold than in normal healthy women (p < 0.05). Haptoglobin expression in the serum of cancer patients (n = 7) decreased following chemotherapy (six cycles of taxol/carboplatin). Concomitant with the decrease of haptoglobin, transferrin expression remained constant in four patients, but increased in three out of seven patients included in the study. Changes in serum expression of haptoglobin correlated with the change of CA 125 levels before and after chemotherapy. In conclusion, proteomic profiling of differentially expressed proteins in the sera of normal women compared to women with ovarian cancer can greatly facilitate the discovery of a panel of biomarkers that may aid in the detection of ovarian cancer with greater specificity.

Amino Acid Sequence↗
PubMed2001

From genomics to proteomics: techniques and applications in cancer research.

New technologies designed to facilitate the comprehensive analyses of genomes, transcriptomes and proteomes in health and disease are poised to exert a dramatic change on the pace of cancer research and to impact significantly on the care of cancer patients. These approaches have already demonstrated the power of molecular medicine in discriminating among disease subtypes that are not recognizable using traditional pathological criteria and in identifying specific genetic events involved in cancer progression. This review outlines the current status of these technologies and highlights recent studies in which they have been applied in the context of carcinogenesis.

Affinity Labels↗
PubMed2005

On-column protein refolding for crystallization.

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an 'artificial chaperone-assisted refolding' method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.

Chromatography, Affinity↗
PubMed2006

Mapping protein-protein interactions by mass spectrometry.

Mass spectrometry is currently at the forefront of technologies for mapping protein-protein interactions, as it is a highly sensitive technique that enables the rapid identification of proteins from a variety of biological samples. When used in combination with affinity purification and/or chemical cross-linking, whole or targeted protein interaction networks can be elucidated. Several methods have recently been introduced that display increased specificity and a reduced occurrence of false-positives. In the future, information gained from human protein interaction studies could lead to the discovery of novel pathway associations and therapeutic targets.

Animals↗
PubMed2005

Comparison of affinity tags for protein purification.

Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.

Affinity Labels↗
PubMed2005

Decrypting the biochemical function of an essential gene from Streptococcus pneumoniae using ThermoFluor technology.

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.

Amino Acid Sequence↗
PubMed2005

Identification of candidate regulators of embryonic stem cell differentiation by comparative phosphoprotein affinity profiling.

Embryonic stem cells are a unique cell population capable both of self-renewal and of differentiation into all tissues in the adult organism. Despite the central importance of these cells, little information is available regarding the intracellular signaling pathways that govern self-renewal or early steps in the differentiation program. Embryonic stem cell growth and differentiation correlates with kinase activities, but with the exception of the JAK/STAT3 pathway, the relevant substrates are unknown. To identify candidate phosphoproteins with potential relevance to embryonic stem cell differentiation, a systems biology approach was used. Proteins were purified using phosphoprotein affinity columns, then separated by two-dimensional gel electrophoresis, and detected by silver stain before being identified by tandem mass spectrometry. By comparing preparations from undifferentiated and differentiating mouse embryonic stem cells, a set of proteins was identified that exhibited altered post-translational modifications that correlated with differentiation state. Evidence for altered post-translational modification included altered gel mobility, altered recovery after affinity purification, and direct mass spectra evidence. Affymetrix microarray analysis indicated that gene expression levels of these same proteins had minimal variability over the same differentiation period. Bioinformatic annotations indicated that this set of proteins is enriched with chromatin remodeling, catabolic, and chaperone functions. This set of candidate phosphoprotein regulators of stem cell differentiation includes products of genes previously noted to be enriched in embryonic stem cells at the mRNA expression level as well as proteins not associated previously with stem cell differentiation status.

Animals↗
PubMed2005

Affinity-tagged phosphorylation assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ATPA-MALDI): application to calcium/calmodulin-dependent protein kinase.

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based kinase assay using a peptide substrate tagged with a biotinyl group has been developed. The peptide moiety was designed to serve as an efficient substrate for calcium/calmodulin-dependent protein kinase II, based on the in vivo phosphorylation site of phosrestin I, a Drosophila homolog of arrestin. In the assay, the quantitative relationship was determined from the ratio of the peak areas between the two peaks respectively representing the unphosphorylated and the phosphorylated substrate. Attempts to assay phosphorylated peptides directly from the reaction mixture, gave inaccurate results because of the high noise level caused by the presence of salts and detergents. In contrast, after purifying the substrate peptides with the biotin affinity tag using streptavidin-coated magnetic beads, peak areas accurately represented the ratio between the unphosphorylated and phosphorylated peptide. By changing the substrate peptide to a peptide sequence that serves as a kinase substrate, it is expected that an efficient non-radioactive protein kinase assay using MALDI-TOF MS can be developed for any type of protein kinase. We call this technique "Affinity-Tagged Phosphorylation Assay by MALDI-TOF MS (ATPA-MALDI)." ATPA-MALDI should serve as a quick and efficient non-radioactive protein kinase assay by MALDI-TOF MS.

Affinity Labels↗
PubMed2006

Chemistry meets proteomics: the use of chemical tagging reactions for MS-based proteomics.

As proteomics matures from a purely descriptive to a function-oriented discipline of the life sciences, there is strong demand for novel methodologies that increase the depth of information that can be obtained from proteomic studies. MS has long played a central role for protein identification and characterization, often in combination with dedicated chemical modification reactions. Today, chemistry is helping to advance the field of proteomics in numerous ways. In this review, we focus on those methodologies that have a significant impact for the large-scale study of proteins and peptides. This includes approaches that allow the introduction of affinity tags for the enrichment of subclasses of peptides or proteins and strategies for in vitro stable isotope labeling for quantification purposes, among others. Particular attention is given to the study of PTMs where recent advancements have been promising, but many interesting targets are not yet being addressed.

Animals↗
PubMed2006

Improvements in the search for potential biomarkers by proteomics: application of principal component and discriminant analyses for two-dimensional maps evaluation.

In this study, we evaluated if the application of multivariate analysis on the data obtained from two-dimensional protein maps could mean an improvement in the search for protein markers. First, we performed a classical proteomic study of the differential expression of serum N-glycoproteins in colorectal cancer patients. Then, applying principal component analysis (PCA) we assessed the utility of the 2-D protein pattern and certain subsets of spots as a tool to distinguish control and case samples, and tested the accuracy of the classification model by linear discriminant analysis (LDA). On the other hand we looked for altered spots by univariate statistics and then analysed them as a cluster by PCA and LDA. We found that those proteins combined presented a theoretical sensitivity and specificity of 100%. Finally, the spots with known protein identity were analysed by multivariate methods, finding a subgroup that behaved as the most obvious candidates for further validation trials.

Biomarkers↗
PubMed2006

Quantitative proteomics identifies a change in glial glutamate metabolism at the time of female puberty.

Mammalian puberty requires activation of luteinizing hormone-releasing hormone (LHRH) neurons. In turn, these neurons are controlled by transsynaptic and glia-to-neuron communication pathways, which employ diverse cellular proteins for proper function. We have now used a high throughput relative quantitative proteomics technique to identify such proteins. We selected the method of two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS) and cleavable isotope-coded affinity tags (cICAT), to both identify and quantify individual proteins within a complex protein mixture. The proteins used derived from the hypothalamus of juvenile (25-day-old) and peripubertal (first proestrus, LP) female rats, and their identity was established by analyzing their mass spectra via database searching. Five proteins involved in glutamate metabolism were detected and two of them appeared to be differentially expressed. They were selected for further analysis, because of their importance in controlling glutamate synthesis and degradation, and their preferential expression in astroglial cells. One, glutamate dehydrogenase (GDH) catalyzes glutamate synthesis; its hypothalamic content detected by 2DLC-MS/MS increases at first proestrus. The other, glutamine synthetase (GS), catalyzes the metabolism of glutamate to glutamine; its content decreases in proestrus. Western blot analysis verified these results. Because these changes suggested an increased glutamate production at puberty, we measured glutamate release from hypothalamic fragments from juvenile 29-day old rats, and from rats treated with PMSG to induce a premature proestrus surge of luteinizing hormone (LH). To determine the net output of glutamate in the absence of re-uptake we used the excitatory amino acid transporter (EAAT) inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC). PDC elicited significantly more glutamate- and LHRH-release from the proestrus hypothalamus. Thus, an increase excitatory drive to the LHRH neuronal network provided by glutamatergic inputs of glial origin, is an event contributing to the pubertal activation of LHRH secretion.

Age Factors↗
PubMed2002

Understanding cancer through proteomics.

Proteomics is a rapidly expanding discipline that aims to gain a comprehensive understanding of the expressions, modification, interactions, and regulation of proteins in cells. New high-throughput technologies, such as protein chips and isotope-coded affinity tag peptide labeling, coupled with classic technologies such as two-dimensional gel electrophoresis and mass spectrometry, complement genomic technologies, providing cancer researchers with powerful tools for cancer diagnosis and prognosis and for the identification of targets for therapy.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2024

Determining small RNA-interacting proteomes using endogenously modified tRNA-derived RNAs.

tRNA-derived RNAs (tDRs), resulting from enzyme-mediated hydrolysis of tRNAs, have been implicated as active small RNAs in various molecular processes. While the molecular modes of action for these small RNAs remain unclear, attempts to decipher the mechanistic details of tDR functionality have mostly used synthetic tDR sequences. Since parental tRNAs are extensively post-transcriptionally modified, tDR functionality is likely affected by chemical modifications. To help approach the biological function of endogenously modified tDRs, this contribution details a protocol that allows purifying specific tDRs carrying post-transcriptional modifications from both in vivo and in vitro sources. Purified tDRs can be used for various downstream applications including differential affinity capture of tDR-binding proteins, the details of which are also described in this contribution.

RNA, Transfer↗
PubMed2026

Multidimensional Protein Corona Analysis Toward Predictive Nano-Bio Interface Design.

Nanoparticles entering biological fluids are rapidly coated by proteins and other biomolecules, converting their synthetic surfaces into biologically active nano-bio interfaces. These coronas regulate colloidal stability, immune recognition, cellular uptake, biodistribution, pharmacokinetics, cargo delivery, and toxicity. Yet a protein list obtained by mass spectrometry captures only part of this interface. Corona identity and function are also shaped by protein organization, binding stability, exchange dynamics, conformational changes, and molecular accessibility. Here, we discuss recent progress in protein corona isolation and analysis from a question-oriented analytical perspective, with emphasis on how centrifugation, magnetic recovery, affinity- or chemistry-enabled capture, chromatography, filtration, and field-flow fractionation (FFF) influence the fidelity, integrity, and comparability of recovered coronas. We then examine how proteomic profiling can be integrated with binding measurements, interfacial structural analysis and functional validation to distinguish descriptive corona signatures from biologically meaningful mechanisms. We further consider how biofluid composition, disease state, tissue interfaces and cellular environments remodel corona identity, presentation, and bioactivity. Finally, we argue that standardized reporting, computational modeling, and AI-enabled approaches are essential for converting protein corona datasets into reproducible and predictive knowledge that can guide the design of drug delivery systems and precision nanomedicines.

Protein Corona↗
PubMed2005

Cross-linking chemistry and biology: development of multifunctional photoaffinity probes.

An efficient method of photoaffinity labeling has been developed based on rationally designed multifunctional photoprobes. Photoaffinity techniques have been used to elucidate the protein structure at the interface of biomolecules by the photochemical labeling of interacting sites. However, the identification of labeled sites within target proteins is often difficult. Novel biotinyl bioprobes bearing a diazirine photophore have contributed significantly to the rapid elucidation of ligand binding sites within proteins, thereby extending conventional photoaffinity methods. This article discusses the synthesis and applications of various photoprobes bearing a biotin, including strategies using cleavable linkages between photophores. The combination of photoaffinity methods with chip technology is also described as a novel entry to rapid affinity-based screening of inhibitors. This review focuses on a rapid and reliable photoaffinity method utilizing diazirine-based multifunctional photoprobes with numerous potential applications in functional proteomics of biomolecular interactions.

Diazomethane↗
PubMed2003

Identification of ATP-synthase as a novel intracellular target for microcystin-LR.

Microcystins (MCs) are a group of closely related cyclic heptapeptides produced by a variety of common cyanobacteria. These are potent and highly specific hepatotoxins, the toxicity of which is based upon their inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases. Apart from protein phosphatases, it is not known whether these phosphatase-inhibiting peptides could bind any other cellular proteins. We wanted to determine whether any possible unknown MC-adducts could explain the apoptotic effects observed at high concentrations of MCs. The question of other possible cellular proteins binding to MCs is also relevant when these compounds are employed for affinity purification of protein phosphatases. In MC-treated cell lysates, antibodies to MC recognized three protein adducts of 35-37 and 55 kD. By immunochemical and proteomics approaches, these proteins were identified as the catalytic subunits of type-1 and type-2A protein phosphatases and the ATP-synthase beta-subunit. The latter target could be associated with the suggested apoptosis-inducing potential of MCs.

Amino Acid Sequence↗
PubMed2006

Proteomic analysis of rat hippocampal plasma membrane: characterization of potential neuronal-specific plasma membrane proteins.

The hippocampus is a distinct brain structure that is crucial in memory storage and retrieval. To identify comprehensively proteins of hippocampal plasma membrane (PM) and detect the neuronal-specific PM proteins, we performed a proteomic analysis of rat hippocampus PM using the following three technical strategies. First, proteins of the PM were purified by differential and density-gradient centrifugation from hippocampal tissue and separated by one-dimensional electophoresis, digested with trypsin and analyzed by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS). Second, the tryptic peptide mixture from PMs purified from hippocampal tissue using the centrifugation method was analyzed by liquid chromatography ion-trap ESI-MS/MS. Finally, the PM proteins from primary hippocampal neurons purified by a biotin-directed affinity technique were separated by one-dimensional electrophoresis, digested with trypsin and analyzed by ESI-Q-TOF-MS/MS. A total of 345, 452 and 336 non-redundant proteins were identified by each technical procedure respectively. There was a total of 867 non-redundant protein entries, of which 64.9% are integral membrane or membrane-associated proteins. One hundred and eighty-one proteins were detected only in the primary neurons and could be regarded as neuronal PM marker candidates. We also found some hypothetical proteins with no functional annotations that were first found in the hippocampal PM. This work will pave the way for further elucidation of the mechanisms of hippocampal function.

Animals↗