Peptide Probe Research Articles

Method Development for Reversed-Phase Separations of Peptides: A Rational Screening Strategy for Column and Mobile Phase Combinations with Complementary Selectivity

This review article summarizes the results obtained from the combined efforts of a joint academic and industrial initiative to solve the real-life challenge of determining low levels of peptide-related impurities (typically 0.05–1% of the drug substance) in the presence of the related biologically active peptide at a high concentration. A rational screening strategy for pharmaceutically important peptides has been developed that uses combinations of reversed‑phase ultrahigh-pressure liquid chromatography (UHPLC) columns and mobile phases that exhibit complementary reversed-phase chromatographic selectivity using either UV- or mass spectrometry (MS)-compatible conditions. Numerous stationary and mobile phases were categorized using the chemometric tool of principal component analysis (PCA), employing a novel characterization protocol utilizing specifically designed peptide probes. This was successfully applied to the development of a strategy for the detection of impurities (especially isomers) in peptide drug substances using two-dimensional liquid chromatography coupled with MS detection (2D-LC–MS).

Reduced graphene oxide quenched peptide probe for caspase-8 activity detection and cellular imaging.

Cysteinyl aspartate-specific protease 8 (caspase-8) plays a key role in various biological processes by regulating apoptosis. Therefore, this makes accurate detection and intracellular imaging of caspase-8 of great importance for drug screening, disease diagnosis, and prognostication. Here, by designing a reduced graphene oxide (rGO) quenched peptide probe, we constructed a new biosensing system for monitoring in vitro and intracellular caspase-8 activity. In this system, a fluorophore-labeled peptide and rGO were used as the substrate of caspase-8 and the fluorophore quencher, respectively. The hydrolysis of caspase-8 on the polypeptide probe substrate can generate two fragments with different lengths. The release of the short fragment labeled with the fluorophore causes recovery of the fluorescence signal (Ex/Em = 520/576nm). Under the optimized conditions, the proposed fluorescence method exhibited a linear response range of 0.2 to 5 U·mL-1 for caspase-8 with a limit of detection (LOD) of 0.2 U·mL-1 in vitro. Furthermore, this method has been successfully applied to monitoring the upregulation of intracellular caspase-8 activity caused by tert-butyl hydroperoxide (TBHP) and fluorouracil. Flow cytometry assay indicated the positive relation between the upregulation of intracellular caspase-8 activity and cell apoptosis rate. In summary, the above results demonstrated the practical application of this method for apoptosis-related cell imaging.

Peptide-based sensing of Pb2+, molecular logic computing, information encoding, cryptography, and steganography

Peptides are a class of powerful molecules and building blocks similar to DNA. However, peptides with composition diversity and molecular recognition ability are rarely used for multipurpose integrated applications (especially in sensing, information processing, communication, and security). Herein, peptide-based sensing of Pb2+, logic computing, information encoding, and security applications were comprehensively demonstrated. Our fluorescently labeled Pb2+-binding peptide exhibited different responses to various metal ions but was specific to Pb2+, which were attributed to the fluorescence static quenching and the transformation of the peptide conformation induced by Pb2+. This peptide probe was successfully used to quantitatively detect Pb2+ (detection limit 3.38 nM) in actual water samples and to perform logic calculations. In addition, due to natural concealment of molecules, encoding, encryption, and hiding of 2 kinds of dimensional information (such as text and images), were displayed by using peptide sequences and its selectivity. This information encoding method based on molecules and their subsidiary properties shows great flexibility and scalability and provides a new option for information representation and protection. Inspired by this idea, more interesting and creative molecular sensing and information systems will be developed to promote connectivity, programmability, and intelligence of the molecular world.

Peptide-anchored biomimetic interface for electrochemical detection of cardiomyocyte-derived extracellular vesicles.

Cardiomyocyte-derived extracellular vesicles (EVs) are a promising class of biomarkers that can advance the diagnosis of many kinds of cardiovascular diseases. Herein, we develop a new electrochemical method for the feasible detection of cardiomyocyte-derived EVs in biological fluids. The core design of the method is the fabrication of a peptide-anchored biomimetic interface consisting of a lipid bilayer and peptide probes. On the one hand, the lipid bilayer provides excellent antifouling ability to the electrode interface and facilitates the anchoring of peptide probes. On the other hand, the peptide probes equip the electrode interface with excellent binding capability and affinity to CD172a, a specific marker of cardiomyocyte-derived EVs, thus enabling the efficient and selective detection of target EVs. Taking EVs derived from the heart myoblast cells H9C2 as the model target, the method displays a wide linear detection range from 1 × 103 to 1 × 108 particles/mL with a desirable detection limit of 132 particles/mL. Furthermore, the method shows good performance in biological fluids such as serum, and thus may have great potential for practical use in the diagnosis of cardiovascular diseases.

Threonine Phosphorylation of an Electrochemical Peptide-Based Sensor to Achieve Improved Uranyl Ion Binding Affinity

We have successfully designed a uranyl ion (U(VI)-specific peptide and used it in the fabrication of an electrochemical sensor. The 12-amino acid peptide sequence, (n) DKDGDGYIpTAAE (c), originates from calmodulin, a Ca(II)-binding protein, and contains a phosphothreonine that enhances the sequence's affinity for U(VI) over Ca(II). The sensing mechanism of this U(VI) sensor is similar to other electrochemical peptide-based sensors, which relies on the change in the flexibility of the peptide probe upon interacting with the target. The sensor was systematically characterized using alternating current voltammetry (ACV) and cyclic voltammetry. Its limit of detection was 50 nM, which is lower than the United States Environmental Protection Agency maximum contaminant level for uranium. The signal saturation time was ~40 min. In addition, it showed minimal cross-reactivity when tested against nine different metal ions, including Ca(II), Mg(II), Pb(II), Hg(II), Cu(II), Fe(II), Zn(II), Cd(II), and Cr(VI). Its reusability and ability to function in diluted aquifer and drinking water samples were further confirmed and validated. The response of the sensor fabricated with the same peptide sequence but with a nonphosphorylated threonine was also analyzed, substantiating the positive effects of threonine phosphorylation on U(VI) binding. This study places emphasis on strategic utilization of non-standard amino acids in the design of metal ion-chelating peptides, which will further diversify the types of peptide recognition elements available for metal ion sensing applications.

A CHANGE OF HEART: A CASE OF CARDIAC AMYLOIDOSIS

A CHANGE OF HEART: A CASE OF CARDIAC AMYLOIDOSIS

HIV-1 gp120-CXCR4 recognition probed with synthetic nanomolar affinity D-peptides containing fragments of gp120 V3 loop

The human immunodeficiency virus type 1 (HIV-1) recognizes one of its principal coreceptors, the CXC chemokine receptor 4 (CXCR4) on the host cell via the third variable loop (V3 loop) of HIV-1 envelope glycoprotein gp120 during the viral entry process. Here, we investigated the stereochemical mechanism of the molecular recognition of HIV-1 gp120 V3 loop with coreceptor CXCR4 by using peptide probes containing important fragments of the V3 loop. The tip and base/stem fragments of the V3 loop critical for V3 loop function were linked individually with the fragment derived from another CXCR4's chemokine ligand, vMIP-II to generate nanomolar affinity peptide probes of the interactions of CXCR4-V3 loop fragments. When the amino acid residues of the V3 loop fragments in these combinational peptides were changed from L-to D-configurations, the resulting peptides remarkably retained or had even enhanced recognition by CXCR4 as shown by competitive ligand-receptor binding. The ability of these peptides, regardless of the different l- or d-amino acids used, in binding CXCR4 and antagonizing CXCR4 functions was demonstrated by their blockade of calcium influx, cell migration, and CXCR4 internalization triggered by the activation of CXCR4 signaling by its endogenous ligand SDF-1α. The structural mechanisms of CXCR4 interactions with these peptides were examined with site-directed mutagenesis and molecular modeling. These results indicate that CXCR4's interface with key segments of HIV-1 gp120 V3 loop is flexible in terms of stereospecificity of ligand-receptor interaction which may have implication on understanding the viral entry mechanism and how the virus evades immune detection with V3 loop mutations and retains effective recognition of the host cell's coreceptor.

Plasmon-Mediated Peroxidase-like Activity on an Asymmetric Nanotube Architecture for Rapid Visual Detection of Bacteria.

Rapid and sensitive detection of bacteria from a complex real media remains a challenge. Herein, we report a visual bacterial sensing assay with excellent specificity, anti-interference ability, and sensitivity based on a surface plasmon resonance (SPR)-enhanced peroxidase (POD) mimetic. The POD mimetic based on Pt nanoparticles (NPs) asymmetrically decorated on Au/TiO2 magnetic nanotubes (Au/Pt/MTNTs) is designed by combining the intrinsic photocatalytic activity of TiO2 and the limited transport depth of light. It is revealed that the localized surface plasmon resonance (LSPR) effect of the asymmetric nanotubes is more effective in facilitating the generation of hot electrons, which are subsequently transferred to Pt and MTNTs, thus greatly promoting the catalytic performance. Using Staphylococcus aureus (S.aureus) as a model of Gram-positive bacteria, the dependence of the colorimetric reaction on the active sites of the POD mimetic is used for the sensing of target bacteria. Owing to the specific recognition between S.aureus and peptide, the fluorescein isothiocyanate (FITC) labeled peptide probes are captured by S.aureus and removed from the Au/Pt/MTNTs, leading to the recovery of POD-like activity and fluorescence emission of S.aureus. Particularly, benefiting from the Au-SPR effect and the magnetic feature of the Au/Pt/MTNTs, the recovery of catalytic activity induced an improved colorimetric assay with a wider linear response for S.aureus qualification and a detection limit of four cells, as well as satisfactory selectivity and feasibility for application in real samples. The plasmon-enhanced POD activity would provide a simple-yet-effective approach to achieve a colorimetric bioassay with high efficiency and sensitivity. This asymmetric design can also be utilized to engineer nanozymes in colorimetric assays for the specific detection of biotoxins, biomarkers, and cancer cells.

Hydrophobicity is a key determinant in the activity of arginine-rich cell penetrating peptides

To deliver useful biological payloads into the cytosolic space of cells, cell-penetrating peptides have to cross biological membranes. The molecular features that control or enhance this activity remain unclear. Herein, a dimeric template of the arginine-rich HIV TAT CPP was used to establish the effect of incorporating groups and residues of various chemical structures and properties. A positive correlation is established between the relative hydrophobicity of these additional moieties and the ability of the CPP conjugates to deliver a peptidic probe into live cells. CPP conjugates with low hydrophobicity lead to no detectable delivery activity, while CPPs containing groups of increasing hydrophobicity achieve intracellular delivery at low micromolar concentrations. Notably, the chemical structures of the hydrophobic groups do not appear to play a role in overall cell penetration activity. The cell penetration activity detected is consistent with endosomal escape. Leakage assays with lipid bilayer of endosomal membrane composition also establish a positive correlation between hydrophobicity and membrane permeation. Overall, these results indicate that the presence of a relatively hydrophobic moiety, regardless of structure, is required in a CPP structure to enhance its cell penetration. It also indicates that simple modifications, including fluorophores used for cell imaging or small payloads, modulate the activity of CPPs and that a given CPP-conjugate may be unique in its membrane permeation properties.

Highly Fluorinated Peptide Probes with Enhanced In Vivo Stability for 19 F-MRI.

A labeling strategy for in vivo 19 F-MRI (magnetic resonance imaging) based on highly fluorinated, short hydrophilic peptide probes, is developed. As dual-purpose probes, they are functionalized further by a fluorophore and an alkyne moiety for bioconjugation. High fluorination is achieved by three perfluoro-tert-butyl groups, introduced into asparagine analogues by chemically stable amide bond linkages. d-amino acids and β-alanine in the sequences endow the peptide probes with low cytotoxicity and high serum stability. This design also yielded unstructured peptides, rendering all 27 19 F substitutions chemically equivalent, giving rise to a single 19 F-NMR resonance with <10Hz linewidth. The resulting performance in 19 F-MRI is demonstrated for six different peptide probes. Using fluorescence microscopy, these probes are found to exhibit high stability and long circulation times in living zebrafish embryos. Furthermore, the probes can be conjugated to bovine serum albumin with only amoderate increase in 19 F-NMR linewidth to ≈30Hz. Overall, these peptide probes are hence suitable for in vivo 19 F-MRI applications.

Development of a protein microarray for profiling circulating autoantibodies in human diseases.

To develop a robust microarray platform to detect thousands of serological autoantibodies (AAbs) simultaneously in different diseases. An AAbMap microarray was prepared by printing a total of 4032 purified His-tagged human proteins and peptide probes on a chemically-modified slide. The sensitivity, dynamic range, and the inter- and intra-array reproducibility of the AAb microarray were then systematically tested and optimized. Finally, the large-scale profiling of AAbs in the serum of patients with different human diseases using the AAbMap microarray was demonstrated. The dynamic range of antibody (Ab) detection was 2 to 3 orders of magnitude with the lowest limit of detection (LOD) of 68 pg/mL. The intra-array (r) correlation of duplicate spots was 1.00, whereas the inter-array correlations between different arrays and batches were 0.99 and 0.97 to 0.98, respectively. Notably, 132, 266, 171, and 84 AAbs were detected in pooled serum from healthy controls (HCs) or patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or lung cancer (LC), respectively. These AAbs included antibodies that target well-known disease biomarkers, such as anti-cyclic citrullinated peptide, anti-ribonucleoprotein, and anti-nucleosome. We developed a microarray platform to measure thousands of serological AAbs simultaneously with high sensitivity and reproducibility. The array can help study autoimmunity and complement genomics, proteomics, and metabolomics data for systematic investigations of human diseases.

Comprehensive analysis of transglutaminase substrate preference by cDNA display coupled with next-generation sequencing and bioinformatics

cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (1012) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions − 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position − 1 (Gln-Gln motif), and isoleucine or valine at position + 3. The highly enriched peptides indeed contained the indicated sequence and showed a higher reactivity as TG2 substrates than the peptide previously selected by phage display, thus representing the novel candidate peptide probes for TG2 research. Furthermore, the obtained information on substrate profiling can be used to identify potential TG2 protein targets. This platform will be further used for the substrate profiling of other TG isozymes, as well as for the selection and evolution of larger biomolecules.

Update on Disease-Specific Biomarkers in Transthyretin Cardiac Amyloidosis.

Transthyretin cardiac amyloidosis (ATTR-CM) is an infiltrative cardiomyopathy and an increasingly recognized cause of morbidity and mortality. There remains substantial delay between initial symptoms and diagnosis. With the recent emergence of various targeted therapies proven to reduce morbidity and mortality, there is an imperative to diagnose subclinical disease. Biomarkers may be well-suited for this role. Conventional markers of heart failure, such as natriuretic peptides and cardiac troponins, and estimated glomerular filtration rate are associated with risk in ATTR-CM. Circulating transthyretin (TTR) levels parallel TTR kinetic stability, correlate with disease severity, and may serve as indirect markers of ATTR-CM disease activity and response to targeted treatment. There is also growing evidence for the correlation of TTR to retinol-binding protein 4, a biomarker which independently associates with this disease. The rate-limiting step for ATTR pathogenesis is dissociation of the TTR homotetramer, which may be quantified using subunit exchange to allow for early risk assessment, prognostication, and assessment of treatment response. The protein species that result from the dissociation and misfolding of TTR are known as nonnative transthyretin (NNTTR). NNTTR is quantifiable via peptide probes and is a specific biomarker whose reduction is positively correlated with improvement in neuropathic ATTR amyloidosis. Neurofilament light chain (NfL) is released into the blood after axonal damage and correlates with neuropathic ATTR amyloidosis, but its clinical use in ATTR-CM is uncertain. Conventional markers of heart failure, transthyretin, retinol-binding protein 4, transthyretin kinetic stability, nonnative transthyretin, peptide probes, and neurofilament light chain have potential as biomarkers to enable early, subclinical diagnosis in patients with transthyretin cardiac amyloidosis.

TUBright: A Peptide Probe for Imaging Microtubules.

In vitro assays using reconstituted microtubules have provided molecular insights into the principles of microtubule dynamics and the roles of microtubule-associated proteins. Emerging questions that further uncover the complexity in microtubule dynamics, especially those on tubulin isotypes and post-translational modifications, raise new technical challenges on how to visualize microtubules composed of tubulin purified from limited sources, primarily due to the low efficiency of the conventional tubulin labeling protocol. Here, we develop a peptide probe, termed TUBright, that labels in vitro reconstituted microtubules. TUBright, when coupled with different fluorescent dyes, provides flexible labeling of microtubules with a high signal-to-noise ratio. TUBright does not interfere with the dynamic behaviors of microtubules and microtubule-associated proteins. Therefore, TUBright is a useful tool for imaging microtubules, making it feasible to use tubulin from limited sources for answering many open questions on microtubule dynamics.

Investigating Peptide-Coenzyme A Conjugates as Chemical Probes for Proteomic Profiling of N-Terminal and Lysine Acetyltransferases.

Acetyl groups are transferred from acetyl‐coenzyme A (Ac‐CoA) to protein N‐termini and lysine side chains by N‐terminal acetyltransferases (NATs) and lysine acetyltransferases (KATs), respectively. Building on lysine‐CoA conjugates as KAT probes, we have synthesized peptide probes with CoA conjugated to N‐terminal alanine (α‐Ala‐CoA), proline (α‐Pro‐CoA) or tri‐glutamic acid (α‐3Glu‐CoA) units for interactome profiling of NAT complexes. The α‐Ala‐CoA probe enriched the majority of NAT catalytic and auxiliary subunits, while a lysine CoA‐conjugate bound only a subset of endogenous KATs. Interactome profiling with the α‐Pro‐CoA probe showed reduced NAT recruitment in favor of metabolic CoA binding proteins and α‐3Glu‐CoA steered the interactome towards NAA80 and NatB. These findings agreed with the inherent substrate specificities of the target proteins and showed that N‐terminal CoA‐conjugated peptides are versatile probes for NAT complex profiling in lysates of physiological and pathological backgrounds.

Endocytosis Pathway Self-Regulation for Precise Image-Guided Therapy through an Enzyme-Responsive Modular Peptide Probe

Before arriving at the intracellular destinations, probes might be trapped in the lysosomes, reducing the amount of cargos, which compromises the therapeutic outcomes. The current methods are based on the fact that probes enter the lysosomes and then escape from them, which do not fundamentally solve the degradation by lysosomal hydrolases. Here, an enzyme-responsive modular peptide probe named PKP that can be divided into two parts, Pal-part and KP-part, by matrix metalloproteinase-2 (MMP-2) overexpressed in tumor microenvironments is designed. Pal-part quickly enters the cells and forms nanofibers in the lysosomes, decreasing protein phosphatase 2A (PP2A), which transforms the endocytic pathway of KP-part from clathrin-mediated endocytosis (CME) into caveolae-mediated endocytosis (CvME) and allows KP-part to directly reach the mitochondria sites without passing through the lysosomes. Finally, through self-regulating intracellular delivery pathways, the mitochondrial delivery efficiency of KP-part is greatly improved, leading to an optimized image-guided therapeutic efficiency. Furthermore, this system also shows great potential for the delivery of siRNA and doxorubicin to achieve precise cancer image-guided therapy, which is expected to significantly expand its application and facilitate the development of personalized therapy.

Efficient synthesis of terminal-diazirine-based histone peptide probes

Histone post-translational modifications (hPTMs) regulate series fundamental cellular processes through the specific recognition of “reader” or “effector” proteins. The chemical probes containing photo-cross-linking groups, especially diazirine group has been developed to capture proteins recognized by hPTMs. However, the construction of diazirine-based photo-reactive amino acids requires multiple synthetic steps. Herein, we report the efficient synthesis of a new diazirine-based histone peptide probes through the construction of terminal diazirine via phenyliodonium diacetate (PIDA) mediated transformation. The key building block tert-butyl 3-(3H-diazirin-3-yl)propanoate can be obtained in one step with 90% yield. The synthetic H3K4Me3 photo-cross-linking probe was demonstrated to label H3K4Me3 specific reader in vitro and in cell lysates.

Rapid high-throughput compatible label-free virus particle quantification method based on time-resolved luminescence

Viruses play a major role in modern society and create risks from global pandemics and bioterrorism to challenges in agriculture. Virus infectivity assays and genome copy number determination methods are often used to obtain information on virus preparations used in diagnostics and vaccine development. However, these methods do not provide information on virus particle count. Current methods to measure the number of viral particles are often cumbersome and require highly purified virus preparations and expensive instrumentation. To tackle these problems, we developed a simple and cost-effective time-resolved luminescence-based method for virus particle quantification. This mix-and-measure technique is based on the recognition of the virus particles by an external Eu3+-peptide probe, providing results on virus count in minutes. The method enables the detection of non-enveloped and enveloped viruses, having over tenfold higher detectability for enveloped, dynamic range from 5E6 to 3E10 vp/mL, than non-enveloped viruses. Multiple non-enveloped and enveloped viruses were used to demonstrate the functionality and robustness of the Protein-Probe method. Graphical abstract

Peptide Ligand Interaction with Maltose‐Binding Protein Tagged to the Calcitonin Gene‐Related Peptide Receptor: Inhibitory Roles of Receptor N‐Glycosylation

Calcitonin gene‐related peptide (CGRP) and adrenomedullin (AM) are peptide hormones and their receptors play a critical role in migraine progression and blood pressure control, respectively. CGRP and AM receptors are structurally related since they are the complex of the calcitonin receptor‐like receptor (CLR) with the different types of receptor activity‐modifying protein (RAMP); the CLR complex with RAMP1 is the CGRP receptor while the CLR complex with RAMP2 is the AM receptor. The extracellular domain (ECD) of CLR and RAMP provides a binding site for peptide ligands as reported in crystal structures. Several crystal structures of the CGRP and AM receptor ECD used maltose‐binding protein (MBP) as a tag protein to facilitate crystallization. Unexpectedly, the recent crystal structures of CGRP receptor ECD showed that the N‐terminal MBP tag located in proximity of the bound/mutated peptide ligands. The objective of the current study is to investigate potential chemical interaction between peptide ligands and the MBP tagged to the CGRP receptor ECD. Peptide ligand interaction with purified CGRP receptor ECD was evaluated with fluorescence polarization/anisotropy peptide binding assay with a fluorescent peptide probe. The results of the current study provided evidence that MBP N‐terminally tagged to the CGRP receptor ECD formed chemical interaction with mutated peptide ligands. Interestingly, N‐glycosylation of the CGRP receptor ECD was predicted to prevent MBP docking to the mutated peptide ligands. The N‐glycosylation of CLR ECD N123 was the most critical for inhibiting MBP interaction with the mutated peptide ligands. The MBP tag protein interaction was also dependent on the sequence of the peptide ligands. An endogenous CGRP fragment did not appear to interact with the MBP tag. In contrast to the CGRP receptor, the MBP tag was not involved in peptide ligand binding at AM receptor ECD. Here, I provided evidence that N‐glycosylation of the CGRP receptor ECD inhibited the MBP tag protein interaction suggesting an additional role of N‐glycosylation in the MBP‐tagged CGRP receptor ECD. This study reveals the importance of using a tag protein‐free CGRP receptor ECD for the accurate assessment of peptide ligand affinity.

O-60 - Development of peptide probes for imaging of CD206 positive macrophages in cancer

O-60 - Development of peptide probes for imaging of CD206 positive macrophages in cancer