Peptide Probe Research Articles

Array-Based Rational Design of Short Peptide Probe-Derived from an Anti-TNT Monoclonal Antibody.

Complementarity-determining regions (CDRs) are sites on the variable chains of antibodies responsible for binding to specific antigens. In this study, a short peptide probe for recognition of 2,4,6-trinitrotoluene (TNT), was identified by testing sequences derived from the CDRs of an anti-TNT monoclonal antibody. The major TNT-binding site in this antibody was identified in the heavy chain CDR3 by antigen docking simulation and confirmed by an immunoassay using a spot-synthesis based peptide array comprising amino acid sequences of six CDRs in the variable region. A peptide derived from heavy chain CDR3 (RGYSSFIYWF) bound to TNT with a dissociation constant of 1.3 μM measured by surface plasmon resonance. Substitution of selected amino acids with basic residues increased TNT binding while substitution with acidic amino acids decreased affinity, an isoleucine to arginine change showed the greatest improvement of 1.8-fold. The ability to create simple peptide binders of volatile organic compounds from sequence information provided by the immune system in the creation of an immune response will be beneficial for sensor developments in the future.

Probing the potential of mucus permeability to signify preterm birth risk

Preterm birth is the leading cause of neonatal mortality, and is frequently associated with intra-amniotic infection hypothesized to arise from bacterial ascension across a dysfunctional cervical mucus plug. To study this dysfunction, we assessed the permeability of cervical mucus from non-pregnant ovulating (n = 20) and high- (n = 9) and low-risk (n = 16) pregnant women to probes of varying sizes and surface chemistries. We found that the motion of negatively charged, carboxylated microspheres in mucus from pregnant patients was significantly restricted compared to ovulating patients, but not significantly different between high- and low-risk pregnant women. In contrast, charged peptide probes small enough to avoid steric interactions, but sensitive to the biochemical modifications of mucus components exhibited significantly different transport profiles through mucus from high- and low-risk patients. Thus, although both microstructural rearrangements of the components of mucus as well as biochemical modifications to their adhesiveness may alter the overall permeability of the cervical mucus plug, our findings suggest that the latter mechanism plays a dominant role in the impairment of the function of this barrier during preterm birth. We expect that these probes may be readily adapted to study the mechanisms underlying disease progression on all mucosal epithelia, including those in the mouth, lungs, and gut.

WS2 and MoS2 biosensing platforms using peptides as probe biomolecules

Biosensors based on the two-dimensional layered nanomaterials transition metal dichalcogenides such as WS2 and MoS2 have shown broad applications, while they largely rely on the utilization of single stranded DNA as probe biomolecules. Herein we have constructed novel WS2- and MoS2- based biosensing platforms using peptides as probe biomolecules. We have revealed for the first time that the WS2 and MoS2 nanosheets display a distinct adsorption for Arg amino acid and particularly, Arg-rich peptdies. We have demonstrated that the WS2 and MoS2 dramatically quench the fluorescence of our constructed Arg-rich probe peptide, while the hybridization of the probe peptide with its target collagen sequence leads to the fluorescence recovery. The WS2-based platform provides a sensitive fluorescence-enhanced assay that is highly specific to the target collagen peptide with little interferences from other proteins. This assay can be applied for quantitative detection of collagen biomarkers in complex biological fluids. The successful development of WS2- and MoS2- based biosensors using non-ssDNA probes opens great opportunities for the construction of novel multifunctional biosensing platforms, which may have great potential in a wide range of biomedical field.

Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide

The development of novel assays to detect collagen fragments is of utmost importance for diagnostic, prognostic and therapeutic decisions in various collagen-related diseases, and one essential question is to discover probe peptides that can specifically recognize target collagen sequences. Herein we have developed the fluorescence self-quenching assay as a convenient tool to screen the capability of a series of fluorescent probe peptides of variable lengths to bind with target collagen peptides. We have revealed that the targeting ability of probe peptides is length-dependent, and have discovered a relatively short probe peptide FAM-G(POG)8 capable to identify the target peptide. We have further demonstrated that fluorescence self-quenching assay together with this short probe peptide can be applied to specifically detect the desired collagen fragment in complex biological media. Fluorescence self-quenching assay provides a powerful new tool to discover effective peptides for the recognition of collagen biomarkers, and it may have great potential to identify probe peptides for various protein biomarkers involved in pathological conditions.

Biscysteine-Bearing Peptide Probes To Reveal Extracellular Thiol-Disulfide Exchange Reactions Promoting Cellular Uptake.

In recent years, delivery systems based on the incorporation of thiols/disulfides have been extensively explored to promote the intracellular delivery of biological cargoes. However, it remains unclear about the detailed processes of thiol-disulfide exchanges taking place on the cell surface and how the exchange reactions promote the cellular uptake of cargoes bearing thiols or disulfide bonds. In this work, we report the rational design of biscysteine motif-containing peptide probes with substantially different ring-closing property and how these peptide probes were employed to explore the thiol-disulfide exchanges on the cell surface. Our results show that extensive thiol-disulfide exchanges between peptides and exofacial protein thiols/disulfides are involved in the cellular uptake of these peptide probes, and importantly glutathione (GSH) exported from the cytosols participates extensively in the exchange reactions. Cysteine-glycine-cysteine (CGC)-containing peptide probes can be more efficiently taken up by cells compared to other probes, and we suggested that the driving force for the superior cellular uptake arises from very likely the unique propensity of the CGC motif in forming doubly bridged disulfide bonds with exofacial proteins. Our probe-based strategy provides firsthand information on the detailed processes of the exchange reactions, which would be of great benefit to the development of delivery systems based on the extracellular thiol-disulfide exchanges for intracellular delivery of biologics.

Designing Probes for Immunodiagnostics: Structural Insights into an Epitope Targeting Burkholderia Infections

Structure-based epitope prediction drives the design of diagnostic peptidic probes to reveal specific antibodies elicited in response to infections. We previously identified a highly immunoreactive epitope from the peptidoglycan-associated lipoprotein (Pal) antigen from Burkholderia pseudomallei, which could also diagnose Burkholderia cepacia infections. Here, considering the high phylogenetic conservation within Burkholderia species, we ask whether cross-reactivity can be reciprocally displayed by the synthetic epitope from B. cenocepacia. We perform comparative analyses of the conformational preferences and diagnostic performances of the corresponding epitopes from the two Burkholderia species when presented in the context of the full-length proteins or as isolated peptides. The effects of conformation on the diagnostic potential and cross-reactivity of Pal peptide epitopes are rationalized on the basis of the 1.8 Å crystal structure of B. cenocepacia Pal and through computational analyses. Our results are discussed in the context of designing new diagnostic molecules for the early detection of infectious diseases.

A strategy for actualization of active targeting nanomedicine practically functioning in a living body.

Designing nanocarriers with active targeting has been increasingly emphasized as for an ideal delivery mechanism of anti-cancer therapeutic agents, but the actualization has been constrained by lack of reliable strategy ultimately applicable. Here, we designed and verified a strategy to achieve active targeting nanomedicine that works in a living body, utilizing animal models bearing a patient's tumor tissue and subjected to the same treatments that would be used in the clinic. The concept for this strategy was that a novel peptide probe and its counterpart protein, which responded to a therapy, were identified, and then the inherent ability of the peptide to target the designated tumor protein was used for active targeting invivo. An initial dose of ionizing radiation was locally delivered to the gastric cancer (GC) tumor of a patient-derived xenograft mouse model, and phage-displayed peptide library was intravenously injected. The peptides tightly bound to the tumor were recovered, and the counterpart protein was subsequently identified. Peptide-conjugated liposomal drug showed dramatically improved therapeutic efficacy and possibility of diagnostic imaging with radiation. These results strongly suggested the potential of our strategy to achieve invivo functional active targeting and to be applied clinically for human cancer treatment.

Peptides derived from MARCKS block coagulation complex assembly on phosphatidylserine

Blood coagulation involves activation of platelets and coagulation factors. At the interface of these two processes resides the lipid phosphatidylserine. Activated platelets expose phosphatidylserine on their outer membrane leaflet and activated clotting factors assemble into enzymatically active complexes on the exposed lipid, ultimately leading to the formation of fibrin. Here, we describe how small peptide and peptidomimetic probes derived from the lipid binding domain of the protein myristoylated alanine-rich C-kinase substrate (MARCKS) bind to phosphatidylserine exposed on activated platelets and thereby inhibit fibrin formation. The MARCKS peptides antagonize the binding of factor Xa to phosphatidylserine and inhibit the enzymatic activity of prothrombinase. In whole blood under flow, the MARCKS peptides colocalize with, and inhibit fibrin cross-linking, of adherent platelets. In vivo, we find that the MARCKS peptides circulate to remote injuries and bind to activated platelets in the inner core of developing thrombi.

Design of carbohydrate/electron-transfer peptides for human histocytic lymphoma cell sensing

A carbohydrate/electro-transfer peptide probe was fabricated to perform cell sensing. The probe consisted of a cello-oligosaccharide that was created by the conjugation of an electron-transfer peptide (Y5C) and a carbohydrate via a Schiff base. An oxidation wave due to a phenolic hydroxyl group was obtained by scanning with a glassy carbon electrode. This cell-sensing system was based on a competitive reaction between carbohydrates on a cell surface and the probe as each reacted to a protein that recognized the carbohydrate. When amounts of the protein and probe were constant, the peak current of the probe was changed as the number of cells increased. A human histocytic lymphoma cell (U937 cell) with carbohydrates such as glucose and N-acetylglucosamine on its surface was selected as the target cell. Wheat germ agglutinin (WGA) binded to both the probe and the carbohydrates on U937 cells, which resulted in a linear peak current of the cellobiose/electron-transfer peptide at concentrations that ranged from 100 to 3500 cells/ml. The values of the cell sensing using this electrochemical method were consistent with those established via ELSIA. The sensitivity of this procedure, however, was two-fold superior to that of ELISA. Consequently, this carbohydrate/electron-transfer peptide could be a powerful tool for cell sensing and searching for carbohydrate chains on a cell surface.

Turning Nonspecific Interference into Signal Amplification: Covalent Biosensing Nanoassembly Enabled by Metal-Catalyzed Cross-Coupling.

In this work, a new method of protein detection in complicated samples is proposed. This method employs probe-target recognition to induce cross-linking among the probe, the target, and the nonspecific proteins in the complicated sample as a means to convert interference into effective signal amplification. This also eliminates the necessity of multistep signal amplification in a separate solution system. On the basis of this strategy, a simple and robust assay for the activity of serum cathepsin B is established. Peptide probes immobilized on a sensing slide can recognize cathepsin B, and this can induce thiol-alkyne covalent coupling between the probe and cathepsin B. Meanwhile, applying electrochemical potential scanning to this sensing surface, Cu binding fragments of the probe peptide can be released into the solution phase to act as an electrochemical catalyst for oxidative dityrosine cross-linking among all proteins including the captured cathepsin B and the nonspecific proteins. A continuous nanoassembly covalently anchored on the sensing surface can gradually form, allowing violent detergent rinsing to remove residual interference. Using this method, not only sensitivity in the picomolar range can be achieved for serum analysis, the results of the analysis can also reliably discriminate benign and cancerous ovarian conditions. These results may suggest prospective application of this method in early screening of cancer in the future.

Peptide-Mediated Nanopore Detection of Uranyl Ions in Aqueous Media.

Uranium is one of the most common radioactive contaminants in the environment. As a major nuclear material in production, environmental samples (like soil and groundwater) can provide signatures on uranium production activity inside the facility. Thus, developing a new and portable analytical technology for uranium in aqueous media is significant not only for environmental monitoring, but also for nonproliferation. In this work, a label-free method for the detection of uranyl (UO22+) ions is developed by monitoring the translocation of a peptide probe in a nanopore. Based on the difference in the number of peptide events in the absence and presence of uranyl ions, nanomolar concentration of UO22+ ions could be detected in minutes. The method is highly selective; micromolar concentrations of Cd2+, Cu2+, Zn2+, Ni2+, Pb2+, Hg2+, Th4+, Mg2+, and Ca2+ would not interfere with the detection of UO22+ ions. In addition, simulated water samples were successfully analyzed.

Electrochemical Peptide-Based (E-PB) Sensor for the Detection of Uranium

Metal ion sensing attracts significant attention as many metal ions that are introduced into natural waters as water waste from industrial processes are toxic to humans. Uranium is a radioactive heavy metal naturally present in the environment and largely found in its hexavalent oxidation state as the uranyl ion, UO2 2+. Anthropogenic activities such as mining, milling, and nuclear testing increase the distribution of groundwater contamination and in many regions in the United States contamination exceeds the U.S. Environmental Protection Agency (EPA) Maximum Contamination Level (MCL) of 30 µg/L (126 nM). Therefore, there is a need for developing sensitive, selective, and cost-effective analytical methods capable of identifying and quantifying uranium in environmental samples. In this study, we focus on understanding the interactions between uranium-binding peptides and UO2 2+ as well as exploiting these interactions in the design of electrochemical peptide-based (E-PB) uranium sensors. We are particularly interested in assessing the effect of threonine phosphorylation on uranium binding affinity of peptides. The peptide probe used in the fabrication of the first generation E-PB uranium sensor is a thiolated 12-amino acid peptide probe modified with methylene blue (MB). In the absence of the target, the probe is flexible and the MB current is high; in the presence of the target, the flexibility of the probe is dampened, resulting in a large reduction in the MB current. Our preliminary results show that the sensor is capable of specific detection of UO2 2+ at concentrations substantially lower than the EPA MCL (limit of detection = 10 nM). Like most E-PB sensors, it is also reusable and selective enough to be employed in 50% synthetic aquifer samples. With further optimization, this sensor could find applications in real time detection of uranium in complex environmental samples.

Peptide probes derived from pertuzumab by molecular dynamics modeling for HER2 positive tumor imaging.

A high level of HER2 expression in breast cancer correlates with a higher tumor growth rate, high metastatic potential, and a poor long-term patient survival rate. Pertuzumab, a human monoclonal antibody, can reduce the effect of HER2 overexpression by preventing HER2 dimerization. In this study, a combination protocol of molecular dynamics modeling and MM/GBSA binding free energy calculations was applied to design peptides that interact with HER2 based on the HER2/pertuzumab crystal structure. Based on a β hairpin in pertuzumab from Glu46 to Lys65—which plays a key role in interacting with HER2—mutations were carried out in silico to improve the binding free energy of the hairpin that interacts with the Phe256-Lys314 of the HER2 protein. Combined the use of one-bead-one-compound library screening, among all the mutations, a peptide (58F63Y) with the lowest binding free energy was confirmed experimentally to have the highest affinity, and it may be used as a new probe in diagnosing and treating HER2-positive breast cancer.

Dual-targeting peptide probe for sequence- and structure-sensitive sensing of serum albumin

Peptide-protein interactions mediate numerous biologic processes and provide great opportunity for developing peptide probes and analytical approaches for detecting and interfering with recognition events. Molecular interactions usually take place on the heterogeneous surface of proteins, and the spatial distribution and arrangement of probes are therefore crucial for achieving high specificity and sensitivity in the bioassays. In this study, small linear peptides, homogenous peptide dimers and hetero bivalent peptides were designed for site-specific recognition of human serum albumin (HSA). Three hydrophilic regions located at different subdomains of HSA were chosen as targets for the molecular design. The binding affinity, selectivity and kinetics of the candidates were screened with surface plasmon resonance imaging (SPRi) and fluoroimmuno assays. Benefiting from the synergistic effect from the surface-targeted peptide binders and the flexible spacer, a heterogenetic dimer peptide (heter-7) with fast binding and slow dissociation behavior was identified as the optimized probe. Heter-7 specifically recognizes the target protein HSA, and effectively blocks the binding of antibody to HSA. Its inhibitory activity was estimated as 83nM. It is noteworthy that heter-7 can distinguish serum albumins from different species despite high similarities in sequence and structure of these proteins. This hetero bivalent peptide shows promise for use in serum proteomics, disease detection and drug transport, and provides an effective approach for promoting the affinity and selectivity of ligands to achieve desirable chemical and biological outcomes.

Tu1962 - Novel Peptide Probe Targets GPC3 Overexpression in Vivo in Hepatocellular Carcinoma

Tu1962 - Novel Peptide Probe Targets GPC3 Overexpression in Vivo in Hepatocellular Carcinoma

Photocontrolled SiRNA Delivery and Biomarker-Triggered Luminogens of Aggregation-Induced Emission by Up-Conversion NaYF4:Yb3+Tm3+@SiO2 Nanoparticles for Inducing and Monitoring Stem-Cell Differentiation.

Controlling the differentiation of stem cells and monitoring cell differentiation has attracted much research interest since the discovery of stem cells. In this regard, a novel near-infrared (NIR) light-activated nanoplatform is obtained by encapsulating the photoactivatable caged compound (DMNPE/siRNA) and combining a MMP13 cleaved imaging peptide-tetrapheny-lethene (TPE) unit conjugated with the mesoporous silica-coated up-conversion nanoparticles (UCNPs) for the remote control of cell differentiation and, simultaneously, for the real-time monitoring of differentiation. Upon NIR light illumination, the photoactivated caged compound is activated, and the siRNA is released from UCNPs, allowing controlled differentiation of stem cells by light. More importantly, MMP13 enzyme triggered by osteogenic differentiation would effectively cleave the TPE probe peptide, thereby allowing the real-time monitoring of differentiation in living stem cells by aggregation-induced emission (AIE).

Sonophore labeled RGD: a targeted contrast agent for optoacoustic imaging

Optoacoustic imaging is a rapidly expanding field for the diagnosis, characterization, and treatment evaluation of cancer. However, the availability of tumor specific exogenous contrast agents is still limited. Here, we report on a small targeted contrast agent for optoacoustic imaging using a black hole quencher® (BHQ) dye. The sonophore BHQ-1 exhibited strong, concentration-dependent, optoacoustic signals in phantoms, demonstrating its ideal suitability for optoacoustic imaging. After labeling BHQ-1 with cyclic RGD-peptide, BHQ-1-cRGD specifically bound to αvβ3-integrin expressing glioblastoma cell spheroids in vitro. The excellent optoacoustic properties of BHQ-1-cRGD could furthermore be proven in vivo. Together with this emerging imaging modality, our sonophore labeled small peptide probe offers new possibilities for non-invasive detection of molecular structures with high resolution in vivo and furthers the specificity of optoacoustic imaging. Ultimately, the discovery of tailor-made sonophores might offer new avenues for various molecular optoacoustic imaging applications, similar to what we see with fluorescence imaging.

Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein-Protein Interaction.

Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.

Label-free, Water-soluble Fluorescent Peptide Probe for a Sensitive and Selective Determination of Copper Ions.

This study aimed to develop a label-free, sensitive, selective, and environment-friendly fluorescent peptide probe His-His-Trp-His (HHWH) for determining the concentration of copper ion (Cu2+) in aqueous solutions. The results demonstrated that the designed HHWH has a high selectivity and sensitivity for monitoring the concentration of free Cu2+ via quenching of the probe fluorescence upon a binding of Cu2+. The fluorescence intensity of the HHWH had a linear relationship with the concentration of Cu2+ between 10 nM and 10 μM, and the detection limit was 8 nM. Furthermore, HHWH could be regenerated with sulfide ions at least five times. The concentrations of Cu2+ in three different real water samples were detected using this probe, and the results were consistent with the one detected using an atomic absorption spectrometer. Thus, HHWH can be used as an accurate and feasible fluorescent peptide probe for detecting Cu2+ in aqueous solutions.

Versatile Interacting Peptide (VIP) Tags for Labeling Proteins with Bright Chemical Reporters.

Fluorescence microscopy is an essential tool for the biosciences, enabling the direct observation of proteins in their cellular environment. New methods that facilitate attachment of photostable synthetic fluorophores with genetic specificity are needed to advance the frontiers of biological imaging. Here, we describe a new set of small, selective, genetically encoded tags for proteins based on a heterodimeric coiled-coil interaction between two peptides: CoilY and CoilZ. Proteins expressed as a fusion to CoilZ were selectively labeled with the complementary CoilY fluorescent probe peptide. Fluorophore-labeled target proteins were readily detected in cell lysates with high specificity and sensitivity. We found that these versatile interacting peptide (VIP) tags allowed rapid and specific delivery of bright organic dyes or quantum dots to proteins displayed on living cells. Additionally, we validated that either CoilY or CoilZ could serve as the VIP tag, which enabled us to observe two distinct cell-surface protein targets with this one heterodimeric pair.