Rapid identification of improved protein ligands using peptoid microarrays.
Ontology highlight
ABSTRACT:
SUBMITTER: Lim HS
PROVIDER: S-EPMC4452005 | biostudies-literature | 2009 Jul
REPOSITORIES: biostudies-literature
Publications
Rapid identification of improved protein ligands using peptoid microarrays.
Lim Hyun-Suk HS Reddy M Muralidhar MM Xiao Xiangshu X Wilson Johnnie J Wilson Rosemary R Connell Steven S Kodadek Thomas T
Bioorganic & medicinal chemistry letters 20090405 14
A rapid array-based protocol is presented by which a modest affinity protein-binding small molecule can be appended to a library of peptoids via click chemistry. The array can then be screened for improved ligands that exhibit a higher affinity for the protein target. ...[more]
Similar Datasets
Project description:We have screened peptoid microarrays to identify specific ligands for the RNA hairpin precursor of miR-21, a microRNA involved in cancer and heart disease. Microarrays were printed by spotting a library of 7680 N-substituted oligoglycines (peptoids) onto glass slides. Two compounds on the array specifically bind RNA having the sequence and predicted secondary structure of the miR-21 precursor hairpin and have specific affinity for the target in solution. Their binding induces a conformational change around the hairpin loop, and the most specific compound recognizes the loop sequence and a bulged uridine in the proximal duplex. Functional groups contributing affinity and specificity were identified, and by varying a critical methylpyridine group, a compound with a dissociation constant of 1.9 microM for the miR-21 precursor hairpin and a 20-fold discrimination against a closely-related hairpin was created. This work describes a systematic approach to discovery of ligands for specific pre-defined novel RNA structures. It demonstrates discovery of new ligands for an RNA for which no specific lead compounds were previously known by screening a microarray of small molecules.
| S-EPMC2760809 | biostudies-other
Project description:We report here that microarrays comprised of several thousand peptoids (oligo-N-substituted glycines) are useful tools for the identification of proteins via a "fingerprinting" approach. By using maltose-binding protein, glutathione S-transferase, and ubiquitin, a specific and highly reproducible pattern of binding was observed when fluorescently labeled protein was hybridized to the array. A similar pattern was obtained when binding of an unlabeled protein to the array was visualized by secondary hybridization of a labeled antibody against that protein, showing that native proteins can be identified without the requirement for prior chemical labeling. This work suggests that small-molecule microarrays might be used for more complex fingerprinting assays of potential diagnostic value.
| S-EPMC1200257 | biostudies-literature
Project description:Hexavalent chromium [Cr(VI)] is a worldwide water contaminant that is currently without cost-effective and efficient remediation strategies. This is in part due to a lack of ligands that can bind it amid an excess of innocuous ions in aqueous solution. We present herein the design and application of a peptoid-based library of ligand candidates for toxic metal ions. A selective screening process was used to identify members of the library that can bind to Cr(VI) species at neutral pH and in the presence of a large excess of spectator ions. There were 11 sequences identified, and their affinities were compared using titrations monitored with UV-vis spectroscopy. To identify the interactions involved in coordination and specificity, we evaluated the effects of sequence substitutions and backbone variation in the highest affinity structure. Additional characterization of the complex formed between this sequence and Cr(VI) was performed using NMR spectroscopy. To evaluate the ability of the developed sequences to remediate contaminated solutions, the structures were synthesized on a solid-phase resin and incubated with environmental water samples that contained simulated levels of chromium contamination. The synthetic structures demonstrated the ability to reduce the amount of toxic chromium to levels within the range of the EPA contamination guidelines. In addition to providing some of the first selective ligands for Cr(VI), these studies highlight the promise of peptoid sequences as easily prepared components of environmental remediation materials.
| S-EPMC4136388 | biostudies-literature
Project description:Protein interaction reporter (PIR) technology can enable identification of in vivo protein interactions with the use of specialized chemical cross-linkers, liquid chromatography, and high-resolution mass spectrometry. PIR-cross-linkers contain labile bonds that are specifically fragmented under low energy collision or photodissociation conditions in the mass spectrometer source, thus releasing cross-linked peptides. Successful analysis of PIR-cross-linked proteins requires the use of expected mathematical relationships between cross-linked complexes and released peptides after fragmentation of the labile PIR bonds. Presented here is a next-generation software tool, BLinks, for use in the analysis and identification of PIR-cross-linked proteins. BLinks is an advancement beyond our previous efforts by incorporation of chromatographic profiles that must match between cross-linked complexes and released peptides to enable estimation of p-values to help filter true relationships from complex data sets. Additionally, BLinks was used to incorporate Mascot database searching results from subsequent MS/MS analysis of the released peptides to facilitate identification of cross-linked proteins. BLinks was used in the analysis of human serum albumin, and 46 interpeptide relationships were found spanning 30 proximal residues with a 2.2% false discovery rate. BLinks was also used to track peptides involved in multiple, coeluting relationships that make accurate identification of protein interactions difficult. An additional 10 interpeptide relationships were identified despite poor correlation using the profiling tools provided with BLinks. Additionally, BLinks can be used to globally map all interpeptide relationships from the data analysis and customize subsequent analysis to target specific peptides of interest, thus making it a useful tool for both discovery of protein interactions and mapping protein topology.
| S-EPMC3018735 | biostudies-literature
Project description:Synthetic equivalents of phosphoprotein-specific antibodies would be valuable reagents for biological research, since these antibodies can often be difficult to produce. Protein phosphorylation is thought to result in significant conformational changes in most substrate proteins. Therefore, one approach might be to simply screen combinatorial libraries for ligands to the phosphorylated state in the hope of isolating a ligand that binds to a pocket created by the conformational shift. In this study, we probe this strategy by screening a peptoid library for ligands to the phosphorylated form of the Brd4 chromatin adaptor and transcriptional coactivator protein. We find that peptoids with high selectivity for binding to the phosphorylation form of Brd4 can indeed be isolated in this screen. Moreover, these ligands do not bind promiscuously to other phospho-proteins. However, attempts to employ these reagents as antibody substitutes in an immunoaffinity purification-like application showed that they do not perform as well as bona fide antibodies and that significant optimization will be required. This study highlights the potential and current limitations of a naïve library screening strategy for phosphoprotein-specific antibody surrogates.
| S-EPMC3591469 | biostudies-literature
Project description:A "one bead two compound" approach to the synthesis of encoded cyclic peptoid libraries is reported.
| S-EPMC4467580 | biostudies-literature
Project description:Secretory IgA plays an important role in the mucosal immune system for protection against
| S-EPMC9533030 | biostudies-literature
Project description:Antimicrobial peptides (AMPs) are intensively studied in terms of alternative drugs. Sub5 is a synthetic 12-mer AMP with substitutions of five amino acids of bactenecin 2A (Bac2A), a linear-ized bactenecin variant of bovine. Sub5 is highly effective against fungi with an ability to trans-locate cell membrane, but its targets are unknown. Systematic analysis of Sub5 targets will facil-itate our understanding on its mechanism of action. In this study, we used high-throughput Saccharomyces cerevisiae proteome microarrays to explore the potential protein targets of Sub5. The screening results showed 128 potential protein targets of Sub5. Bioinformatics analysis of protein targets of Sub5 revealed significant gene ontology (GO) enrichment in actin related pro-cess of "actin filament-based process", "actin filament organization", "actin cortical patch or-ganization", regulation of "actin filament bundle assembly". Moreover, the other enriched cat-egories in GO enrichment mostly contained actin associate proteins. In total, 11 actin-associated proteins were identified in the protein targets of Sub5. Protein family (PFAM) enrichment anal-ysis shows protein domain enriched in actin binding, i.e., "Cytoskeletal-regulatory complex EF hand (helix E-loop-helix F motif)". Being consistent with GO analysis, Search Tool for the Re-trieval of Interacting Genes/Proteins (STRING) analysis of the protein targets of Sub5 showed ac-tin network with involvement of 15 protein targets. Along with actin-network, STRING analysis showed protein-protein interaction network in ribonucleoprotein, transcription and translation, chromosome, histone, and ubiquitin related, DNA repair, and chaperone. Multiple Expression motifs for Motif Elicitation (MEME) suite provided a consensus binding motif of [ED][ED]EEE[ED][ED][ED][ED][ED], in total of 75 protein targets of Sub5. This motif was present in 9 out of 15 actin-related proteins identified among protein targets of Sub5.
| S-EPMC7828587 | biostudies-literature
Project description:BACKGROUND:The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays. RESULTS:We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability. CONCLUSIONS:Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions.
| S-EPMC3562209 | biostudies-literature
Project description:Ovarian cancer is a leading cause of deaths, yet many aspects of the biology of the disease and a routine means of its detection are lacking. We have used protein microarrays and autoantibodies from cancer patients to identify proteins that are aberrantly expressed in ovarian tissue. Sera from 30 cancer patients and 30 healthy individuals were used to probe microarrays containing 5,005 human proteins. Ninety-four antigens were identified that exhibited enhanced reactivity from sera in cancer patients relative to control sera. The differential reactivity of four antigens was tested by using immunoblot analysis and tissue microarrays. Lamin A/C, SSRP1, and RALBP1 were found to exhibit increased expression in the cancer tissue relative to controls. The combined signals from multiple antigens proved to be a robust test to identify cancerous ovarian tissue. These antigens were also reactive with tissue from other types of cancer and thus are not specific to ovarian cancer. Overall our studies identified candidate tissue marker proteins for ovarian cancer and demonstrate that protein microarrays provide a powerful approach to identify proteins aberrantly expressed in disease states.
| S-EPMC2077284 | biostudies-literature