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 217 records · Page 12Linked to original sources
PubMed2006

Functional proteomic profiling of glycan-processing enzymes.

Glycoconjugates play critical roles in regulating cellular and organismal functions. Consequently, defining the relative levels of these glycoconjugates under varied physiological conditions is important. Thus identifying and understanding the regulation of the enzymes that process these glycoconjugates are essential steps in understanding the role of this "glycocode" in development and disease. Activity-based affinity reagents are useful tools for probing these enzymes and should facilitate the unraveling of proteomes. One advantage of activity-based affinity probes is that they can simultaneously reveal multiple enzymes having similar activities. These probes can also be used to enrich proteomes of interest, thereby facilitating identification and cloning of new carbohydrate-processing enzymes. Here we review the current state of activity-based affinity probes for profiling carbohydrate-processing enzymes, focusing on successes and limitations, general design features, and a specific example describing profiling of exoglycosidases from cell lysates.

Affinity Labels↗
PubMed2004

Contributions of commercial sorbents to the selectivity in immobilized metal affinity chromatography with Cu(II).

Immobilized copper(II) affinity chromatography [Cu(II)-immobilized metal affinity chromatography (IMAC)] has been used in proteomics to simplify sample mixtures by selecting histidine-containing peptides from proteolytic digests. This paper examines the specificity of four different support materials with an iminodiacetic acid (IDA) stationary phase in the selection of only histidine-containing peptides in the single step capture-release mode. Three of the sorbents examined were commercially available: HiTrap Chelating HP (agarose), TSK Chelate-5PW, and Poros 20MC. IDA was also immobilized on CIM discs (monolithic glycidylmethacrylate-ethylene dimethacrylate). Tryptic digests of transferrin and beta-galactosidase were used as model samples to evaluate these sorbents. It was found that among the examined matrices, the TSK Chelate-5PW sorbent bound histidine-containing peptides the strongest, while Poros matrix was found to have a high degree of non-specific bindings. Agarose-based columns showed relatively high selectivity and specificity.

Absorption↗
PubMed2006

Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads.

Efficient tagging methodologies are an integral aspect of protein complex characterization by proteomic approaches. Owing to the very high affinity of biotin for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of protein complexes. The very high affinity of the biotin/(strept)avidin system also offers the potential for the single-step capture of lower abundance protein complexes, such as transcription factor complexes. The identification of short peptide tags that are efficiently biotinylated by the bacterial BirA biotin ligase led to an approach for the single-step purification of transcription factor complexes by specific in vivo biotinylation tagging. A short sequence tag fused N-terminally to the transcription factor of interest is very efficiently biotinylated by BirA coexpressed in the same cells, as was demonstrated by the tagging of the essential hematopoietic transcription factor GATA-1. The direct binding to streptavidin of biotinylated GATA-1 in nuclear extracts resulted in the single-step capture of the tagged factor and associated proteins, which were eluted and identified by mass spectrometry. This led to the characterization of several distinct GATA-1 complexes with other transcription factors and chromatin remodeling cofactors, which are involved in activation and repression of gene targets. Thus, BirA-mediated tagging is an efficient approach for the direct capture and characterization of transcription factor complexes.

Amino Acid Sequence↗
PubMed2005

Making the most of affinity tags.

Proteins do not naturally lend themselves to high-throughput analysis because of their diverse physiochemical properties. Consequently, affinity tags have become indispensable tools for structural and functional proteomics initiatives. Although originally developed to facilitate the detection and purification of recombinant proteins, in recent years it has become clear that affinity tags can have a positive impact on the yield, solubility and even the folding of their fusion partners. However, no single affinity tag is optimal with respect to all of these parameters; each has its strengths and weaknesses. Therefore, combinatorial tagging might be the only way to harness the full potential of affinity tags in a high-throughput setting.

Animals↗
PubMed2002

Comparative phenotypic analyses of human plasma and urinary retinol binding protein using mass spectrometric immunoassay.

Mass spectrometric immunoassay (MSIA) is a proteomics technology that combines the selectivity of affinity capture with the sensitivity and resolution of mass spectrometric detection. This unique approach allows for intact protein identification therefore is readily capable of discriminating between protein variants, i.e., mutations, posttranslational modifications, and truncations. In this work, MSIA is used in the comparative analyses of retinol binding protein (RBP) from the plasma and urine of a small study population. Detailed RBP profiles were obtained from both biological fluids, resulting in the identification of several catabolic RBP products (present in urine) that have not been previously reported. In addition, comparative analysis of urine samples taken from healthy and renally impaired individuals revealed different breakdown profiles. These results illustrate the use of MSIA for the rapid, sensitive, and accurate profiling of RBP both within and between individuals.

Adult↗
PubMed2006

Inhibition of a spliceosome turnover pathway suppresses splicing defects.

Defects in assembly are suggested to signal the dissociation of faulty splicing complexes. A yeast genetic screen was performed to identify components of the putative discard pathway. Weak mutant alleles of SPP382 (also called NTR1) were found to suppress defects in two proteins required for spliceosome activation, Prp38p and Prp8p. Spp382p is shown necessary for cellular splicing, with premRNA and, for some alleles, excised intron, accumulating after inactivation. Like spp382-1, a mutant allele of AAR2 was identified in this suppressor screen. Like Spp382p, Aar2p has a reported role in spliceosome recycling and is found with Spp382p in a complex recovered with a mutant version of the spliceosomal core protein Prp8p. Possible insight into to the spp382 suppressor phenotype is provided by the observation that defective splicing complexes lacking the 5' exon cleavage intermediate are recovered by a tandem affinity purification-tagged Spp382 derivative. Stringent proteomic and two-hybrid analyses show that Spp382p also interacts with Cwc23p, a DNA J-like protein present in the spliceosome and copurified with the Prp43p DExD/H-box ATPase. Spp382p binds Prp43p and Prp43p requires Spp382p for intron release from the spliceosome. Consistent with a related function in the removal of defective complexes, three prp43 mutants are also shown to suppress splicing defects, with efficiencies inversely proportionate to the measured ATPase activities. These and related genetic data support the existence of a Spp382p-dependent turnover pathway acting on defective spliceosomes.

Adenosine Triphosphatases↗
PubMed2002

The final N-terminal trimming of a subaminoterminal proline-containing HLA class I-restricted antigenic peptide in the cytosol is mediated by two peptidases.

The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities.

Acetylcysteine↗
PubMed2006

Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein.

BACKGROUND: Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. METHODS: Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed. RESULTS: 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. CONCLUSION: ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer.

Biomarkers, Tumor↗
PubMedDate not supplied

Comparative proteomics of glycoproteins based on lectin selection and isotope coding.

Lectins have been widely used in glycan structure analysis. The studies described here exploit this fact to select glycopeptides carrying disease-associated modifications in their oligosaccharides. Coupling lectin affinity selection with recent advances in stable isotope coding for quantitative proteomics allowed a comparative proteomics method to be developed for examining aberrant glycosylation in cancer. Control and experimental samples were individually tryptic digested and differentially coded with stable isotope coding agents before they were mixed and affinity selected with a lectin affinity chromatography column. Glycopeptides carrying an alpha-L-fucose residue were selected with Lotus tetragonolobus agglutinin (LTA) immobilized on a chromatography matrix. Because the oligosaccharides of glycoproteins are generally heterogeneous and often of unknown structure, it was necessary to deglycosylate the selected peptides with PNGase F before they could be compared to sequences in DNA and protein databases. After deglycosylated peptides were transferred to a reversed phase chromatography (RPC) column and fractionated by gradient elution with increasing amounts of acetonitrile. The RPC fractions were then analyzed by both matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS). When this method was applied to a study of lymphosarcoma in canines, it was found that during chemotherapy, a series of fucosylated proteins in the blood of patients decreased in concentration more than 2-fold. Two of the proteins identified, CD44 and E-selectin, are known to be involved in cell adhesion and cancer cell migration. The observed aberrant fucosylation of these proteins is consistent with the hypothesis that CD44 and E-selectin play a key role in metastasis and the spread of cancer cells to remote sites.

Amino Acid Sequence↗
PubMed2001

Profiling serine hydrolase activities in complex proteomes.

Serine hydrolases represent one of the largest and most diverse families of enzymes in higher eukaryotes, comprising numerous proteases, lipases, esterases, and amidases. The activities of many serine hydrolases are tightly regulated by posttranslational mechanisms, limiting the suitability of standard genomics and proteomics methods for the functional characterization of these enzymes. To facilitate the global analysis of serine hydrolase activities in complex proteomes, a biotinylated fluorophosphonate (FP-biotin) was recently synthesized and shown to serve as an activity-based probe for several members of this enzyme family. However, the extent to which FP-biotin reacts with the complete repertoire of active serine hydrolases present in a given proteome remains largely unexplored. Herein, we describe the synthesis and utility of a variant of FP-biotin in which the agent's hydrophobic alkyl chain linker was replaced by a more hydrophilic poly(ethylene glycol) moiety (FP-peg-biotin). When incubated with both soluble and membrane proteomes for extended reaction times, FP-biotin and FP-peg-biotin generated similar "maximal coverage" serine hydrolase activity profiles. However, kinetic analyses revealed that several serine hydrolases reacted at different rates with each FP agent. These rate differences were exploited in studies that used the biotinylated FPs to examine the target selectivity of reversible serine hydrolase inhibitors directly in complex proteomes. Finally, a general method for the avidin-based affinity isolation of FP-biotinylated proteins was developed, permitting the rapid and simultaneous identification of multiple serine peptidases, lipases, and esterases. Collectively, these studies demonstrate that chemical probes such as the biotinylated FPs can greatly accelerate both the functional characterization and molecular identification of active enzymes in complex proteomes.

Animals↗
PubMed2005

A proteomics strategy for the enrichment of receptor-associated complexes.

Multimeric protein complexes are important for cell function and are being identified by proteomics approaches. Enrichment strategies, such as those employing affinity matrices, are required for the characterization of such complexes, for example, those containing growth factor receptors. The receptor for the macrophage lineage growth factor, macrophage-colony stimulating factor (M-CSF or CSF-1), is the tyrosine kinase, c-Fms. There is evidence that the CSF-1 receptor (CSF-1R) forms distinct multimeric complexes involving autophosphorylated tyrosines in its cytoplasmic region; however, these complexes are difficult to identify by immunoprecipitation, making enrichment necessary. We report here the use of a tyrosine-phosphorylated, GST-fusion construct of the entire CSF-1R cytoplasmic region to characterize proteins putatively associating with the activated CSF-1R. Besides signalling molecules known to associate with the receptor or be involved in CSF-1R-dependent signalling, mass spectrometry identified a number of other molecules binding to the construct. So far among these candidate proteins, dynein, claudin and silencer of death domains co-immunoprecipitated with the CSF-1R, suggesting association. This affinity matrix method, using an entire cytoplasmic region, may have relevance for other growth factor receptors.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Proteome-wide profiling of isoniazid targets in Mycobacterium tuberculosis.

Isoniazid (INH) is an essential drug used to treat tuberculosis. The mycobactericidal agents are INH adducts [INH-NAD(P)] of the pyridine nucleotide coenzymes, which are generated in vivo after INH activation and which bind to, and inhibit, essential enzymes. The NADH-dependent enoyl-ACP reductase (InhA) and the NADPH-dependent dihydrofolate reductase (DfrA) have both been shown to be inhibited by INH-NAD(P) adducts with nanomolar affinity. In this paper, we profiled the Mycobacterium tuberculosis proteome using both the INH-NAD and INH-NADP adducts coupled to solid supports and identified, in addition to InhA and DfrA, 16 other proteins that bind these adducts with high affinity. The majority of these are predicted to be pyridine nucleotide-dependent dehydrogenases/reductases. They are involved in many cellular processes, including S-adenosylmethionine-dependent methyl transfer reactions, pyrimidine and valine catabolism, the arginine degradative pathway, proton and potassium transport, stress response, lipid metabolism, and riboflavin biosynthesis. The targeting of multiple enzymes could, thus, account for the pleiotropic effects of, and powerful mycobactericidal properties of, INH.

Antitubercular Agents↗
PubMed2004

Protein interaction networks by proteome peptide scanning.

A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 microM, while it contains as many as 50-80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment), that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are simultaneously analyzed in an array format, the likelihood of each interaction occurring in any given physiological settings can be evaluated. WISE can be easily extended to a variety of protein interaction domains, including those binding to modified peptides, thereby offering a powerful proteomic tool to help completing a full description of the cell interactome.

Animals↗
PubMed2003

Analysis of transglutaminase protein substrates by functional proteomics.

Transglutaminases are calcium-dependent enzymes that catalyze a post-translational modification of proteins through the formation of epsilon -(gamma-glutamyl)lysine bonds. Although specific roles for transglutaminases have been described, recent findings have provided evidence that dysregulation of transglutaminases may contribute to many pathological processes including celiac disease and neurodegenerative diseases. A crucial step in the elucidation of biological and pathological roles of transglutaminases requires the identification of protein substrates. A strategy based on a functional proteomic analysis was set up using two well-characterized biotinylated transglutaminase substrates as affinity probes: 5-(biotinamido)pentylamine and the synthetic biotinylated peptide TVQQEL, the amino- and acyl-donor probes, respectively. A pool of known tissue type transglutaminase protein substrates was selected in order to test the procedure. Results obtained in this paper indicate that the whole strategy can be successfully applied in order to identify transglutaminases protein substrates as well as the amino acid site sensitive toward enzyme activity.

Affinity Labels↗
PubMed2003

Tumor antigens and proteomics from the point of view of the major histocompatibility complex peptides.

The major histocompatibility complex (MHC) peptide repertoire of cancer cells serves both as a source for new tumor antigens for development of cancer immunotherapy and as a rich information resource about the protein content of the cancer cells (their proteome). Thousands of different MHC peptides are normally displayed by each cell, where most of them are derived from different proteins and thus represent most of the cellular proteome. However, in contrast to standard proteomics, which surveys the cellular protein contents, analyses of the MHC peptide repertoire correspond more to the rapidly degrading proteins in the cells (i.e. the transient proteome). MHC peptides can be efficiently purified by affinity chromatography from membranal MHC molecules, or preferably following transfection of vectors for expression of recombinant soluble MHC molecules. The purified peptides are resolved and analyzed by capillary high-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry, and the data are deciphered with new software tools enabling the creation of large databanks of MHC peptides displayed by different cell types and by different MHC haplotypes. These lists of identified MHC peptides can now be used for searching new tumor antigens, and for identification of proteins whose rapid degradation is significant to cancer progression and metastasis. These lists can also be used for identification of new proteins of yet unknown function that are not detected by standard proteomics approaches. This review focuses on the presentation, identification and analysis of MHC peptides significant for cancer immunotherapy. It is also concerned with the aspects of human proteomics observed through large-scale analyses of MHC peptides.

Antigens, Neoplasm↗
PubMed2006

Antibodies for proteomic research: comparison of traditional immunization with recombinant antibody technology.

Antibodies play a pivotal role in studying the expression and function of proteins. Proteomics studies require the generation of specific and high-affinity antibodies against large numbers of proteins. While traditional animal-based antibody generation is laborious, difficult to automate, and therefore less suited to keep up with the requirements of proteomics research, the use of recombinant in vitro antibody technology might offer a solution to this problem. However, it has not been demonstrated yet that such antibodies are at least as useful as conventional antibodies for typical proteomics applications. Here we generated novel recombinant Fab antibody fragments from the naïve HuCAL GOLD library against a number of targets derived from a mouse cDNA library. We compared these antibodies with polyclonal antisera produced against the same targets and show that these recombinant antibodies are useful reagents for typical applications like Western blotting or immunohistochemistry.

Animals↗