Development of clickable active site-directed photoaffinity probes for γ-secretase

We have developed clickable active site-directed photoaffinity probes for γ-secretase which incorporate a photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated conjugation with azide-linked reporter tags for visualization (e.g., TAMRA-azide) or enrichment (e.g., biotin-azide) of labeled proteins. Specifically, we synthesized clickable analogs of L646 (2) and L505 (3) and validated specific labeling to presenilin-1N-terminal fragment (PS1-NTF), the active site aspartyl protease component within the γ-secretase complex. Additionally, we were able to identify signal peptide peptidase (SPP) by Western blot analysis. Furthermore, we analyzed the photo-labeled proteins in an unbiased fashion by click chemistry with TAMRA-azide followed by in-gel fluorescence detection. This approach expands the utility of γ-secretase inhibitor (GSI) photoaffinity probes in that labeled proteins can be tagged with any number of azide-linked reporters groups using a single clickable photoaffinity probe for target pull down and/or fluorescent imaging applications.     

Design, synthesis and characterization of "clickable" 4-anilinoquinazoline kinase inhibitors

Immobilized kinase inhibitors have emerged as powerful reagents for the determination of kinase inhibitor selectivity and for the enrichment of protein targets from cellular lysates. Here, we report the design and synthesis of a set of "clickable" 4-anilinoquinazoline kinase inhibitors. We demonstrate that the attachment of a flexible tether that contains a bio-orthogonal azide functionality does not adversely affect the potency or selectivity of these inhibitors. Furthermore, we demonstrate the utility of these inhibitors through the generation of an affinity matrix for the enrichment of interacting proteins from cellular lysates.     

Preparation and characterization of nitrilotriacetic-acid-terminated self-assembled monolayers on gold surfaces for matrix-assisted laser desorption ionization-time of flight-mass spectrometry analysis of proteins and peptides

On-target affinity capture, enrichment and purification of biomolecules improve detection of specific analytes from complex biological samples in matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. In this paper, we report a simple method for preparation of a self-assembled nitrilotriacetic acid (NTA) monolayer on gold surface which can be used as a MALDI-TOF-MS sample target specifically for recombinant oligohistidine-tagged proteins/peptides and phosphorylated peptides. The NTA functional groups are immobilized to the gold surface via the linkage of 1,8-octanedithiol which forms a self-assembled monolayer on gold. Characterization by X-ray photoelectron spectroscopy and MALDI analysis of the modified surface are described. The chemically modified surface shows strong affinity toward the analytes of interest, which allows effective removal of the common interferences, e.g. salts and detergents, and therefore leads to improved signal/noise ratio and detection limit. The use of the modified surface simplifies the sample preparation for MALDI analysis of these targeted analytes.     

Current based antibodies detection from human serum enhanced by secondary antibodies labelled with gold nanoparticles immobilized in a nanogap

We present the electrical detection of immunoglobulin G (IgGs) from human serum using a nanogap-based biosensor. The detection method is based on the capture of IgGs by a probe immobilized between gold nanoelectrodes of 30-90nm spacing. The captured IgGs are further reacted with secondary antibodies labelled with gold nanoparticles (GNPs). Insertion of GNPs into the nanogap resulted in increasing the conductance through the nanogap. The use of a chip with 90 nanogaps enabled the calculation of a quality factor for the detection which, coupled with a non-linear regression analysis of the data, easily discriminated specific and differential capture of human antibodies by arrayed probes. We obtained a 500-fold higher quality factor with protein A compared to goat anti-murine antibodies. This method can be applied, through these proof-of-concept experiments, to the detection of protein-protein interactions in biological samples.     

Qualitative and quantitative thrombin analysis in serum using aptamers and gold nanoparticles through progressive dilution

It has been a long-standing challenge in bioassay using aptamers and gold nanoparticles to detect disease-related proteins and other substance directly in complex biological samples such as serum. Here we propose a progressive dilution (PD) method to achieve simultaneous qualitative and quantitative analysis of proteins in blood serum without pretreatment of the sample. Above the detection limit, PD has unlimited dynamic range. We demonstrate the PD strategy through the detection of thrombin in fetal bovine serum using the quenching of fluorescence by gold nanoparticles.     

纳米材料在蛋白质分析中的应用

纳米材料因具有易与蛋白质结合而不影响其生化性质,可用于多种中间体的合成,可与酶、抗体结合而提高其性能等独特的优势而在蛋白质分析中得到了广泛的应用,尤其是与生物技术结合后,对纳米材料在蛋白质分离、富集和检测等方面的作用的研究已成为当前的热点。本文综述了纳米金、石墨烯、碳纳米管和碳纳米球在蛋白质分析中的应用,并对其未来的发展前景进行了展望。     

Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases

The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly.     

FASN inhibitor TVB-3166 prevents S-acylation of the spike protein of human coronaviruses

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses mediates host cell entry and is S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes have been reported to post-translationally modify spike proteins; however, strategies to exploit this modification are lacking. Using resin-assisted capture MS, we demonstrate that the spike protein is S-acylated in SARS-CoV-2-infected human and monkey epithelial cells. We further show that increased abundance of the acyltransferase ZDHHC5 associates with increased S-acylation of the spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also found that the S-acylation of the spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also labelled the protein. Yet, we observed that ZDHHC5 was only modified when incubated with alkyne-palmitate, suggesting it has specificity for this acyl-CoA, and that other ZDHHC enzymes may use additional fatty acids to modify the spike protein. Since multiple ZDHHC isoforms may modify the spike protein, we also examined the ability of the FASN inhibitor TVB-3166 to prevent S-acylation of the spike proteins of SARS-CoV-2 and human CoV-229E. We show that treating cells with TVB-3166 inhibited S-acylation of expressed spike proteins and attenuated the ability of SARS-CoV-2 and human CoV-229E to spread in vitro. Our findings further substantiate the necessity of CoV spike protein S-acylation and demonstrate that de novo fatty acid synthesis is critical for the proper S-acylation of the spike protein.     

Immunochromatographic assay of T-2 toxin using labeled anti-species antibodies

A new scheme of immunochromatographic assay was developed for the highly sensitive detection of low-molecular-weight analytes. This scheme includes the following two steps: the formation of complexes of free specific antibodies with an antigen and their detection by anti-species antibodies conjugated to gold nanoparticles as the label. This scheme was tested with mycotoxin T-2 toxin in maize extracts. The use of specific antibodies and a label as two individual components made it possible to independently vary their concentrations with a simultaneous decrease in the detection limit and an increase in the color intensity. The assay did not require additional reagents and manipulations. The instrumental and visual detection limits of the designed test system were 0.1 and 5.0 ng/mL, respectively (2 and 90 ng per gram of analytes), which are two orders of magnitude lower compared to conventional immunochromatography using the same reagents.     

Affinity separation and enrichment methods in proteomic analysis

Protein separation or enrichment is one of the rate-limiting steps in proteomic studies. Specific capture and removal of highly-abundant proteins (HAP) with large sample-handling capacities are in great demand for enabling detection and analysis of low-abundant proteins (LAP). How to grasp and enrich these specific proteins or LAP in complex protein mixtures is also an outstanding challenge for biomarker discovery and validation. In response to these needs, various approaches for removal of HAP or capture of LAP in biological fluids, particularly in plasma or serum, have been developed. Among them, immunoaffinity subtraction methods based upon polyclonal IgY or IgG antibodies have shown to possess unique advantages for proteomic analysis of plasma, serum and other biological samples. In addition, other affinity methods that use recombinant proteins, lectins, peptides, or chemical ligands have also been developed and applied to LAP capture or enrichment. This review discusses in detail the need to put technologies and methods in affinity subtraction or enrichment into a context of proteomic and systems biology as "Separomics" and provides a prospective of affinity-mediated proteomics. Specific products, along with their features, advantages, and disadvantages will also be discussed.     

Gold nanoparticle-assisted protein enrichment and electroelution for biological samples containing low protein concentration--a prelude of gel electrophoresis

Protein enrichment is essential for biological samples that contain low protein concentrations, especially for proteomic studies that require sufficient quantities for subsequent MS analysis. Traditional precipitation methods, however, are limited in the sample volume and protein concentration required to cause efficient precipitations. We showed that gold nanoparticles (Au-NPs) can be easily applied to concentrate proteins from more than 15 mL of human urine, in which the total protein concentration is less than 1.4 ppm. Moreover, Au-NP-aggregated proteins can be directly applied to gel electrophoresis for Au-NP-protein dissociation followed by free protein separation as well as for the subsequent in-gel digestion and protein identification by mass spectrometry. We compared this method with trichloroacetic acid (TCA) precipitation method, one of the most common precipitation methods, and TCA method showed no enrichment effect for protein samples with large volumes (>2 mL) or with low protein concentrations (4 ppm). Therefore, Au-NP aggregation is not only a simple and efficient method for enriching a broad range of proteins, it is also particularly useful for concentrating proteins from a relatively large volume of dilute biological fluids, under which TCA method is ineffective.     

Electrochemical genosensors for biomedical applications based on gold nanoparticles

Two gold nanoparticles-based genomagnetic sensors designs for detection of DNA hybridization are described. Both assays are based on a magnetically induced direct electrochemical detection of gold tags on magnetic graphite-epoxy composite electrodes. The first design is a two strands assay format that consists of the hybridization between a capture DNA strand which is linked with paramagnetic beads and another DNA strand related to BRCA1 breast cancer gene used as a target which is coupled with streptavidin-gold nanoparticles. The second genomagnetic sensor design is a sandwich assay format with more application possibilities. A cystic fibrosis related DNA strand is used as a target and sandwiched between two complementary DNA probes: the first one linked with paramagnetic beads and a second one modified with gold nanoparticles via biotin-streptavidin complexation reactions. The electrochemical detection of gold nanoparticles by differential pulse voltammetry was performed in both cases. The developed genomagnetic sensors provide a reliable discrimination against noncomplementary DNA as well against one and three-base mismatches. Optimization parameters affecting the hybridization and analytical performance of the developed genosensors are shown for genomagnetic assays of DNA sequences related with the breast cancer and cystic fibrosis genes.     

A piezoelectric immunosensor for specific capture and enrichment of viable pathogens by quartz crystal microbalance sensor, followed by detection with antibody-functionalized gold nanoparticles

A sensitive bacteria enrichment and detection system for viable Escherichia coli O157:H7 was developed using a piezoelectric biosensor-quartz crystal microbalance (QCM) with antibody-functionalized gold nanoparticles (AuNPs) used as detection verifiers and amplifiers. In the circulating-flow QCM system, capture antibodies for E. coli O157:H7 were first immobilized onto the QCM chip. The sample containing E. coli O157:H7 was circulated through the system in the presence of 10ml of brain heart infusion (BHI) broth for 18h. The cells of E. coli O157:H7 specifically captured and enriched on the chip surface of the QCM were identified by QCM frequency changes. Listeria monocytogenes and Salmonella Typhimurium were used as negative controls. After bacterial enrichment, detection antibody-functionalized AuNPs were added to enhance the changes in detection signal. The use of BHI enrichment further enhanced the sensitivity of the developed system, achieving a detection limit of 0-1log CFU/ml or g. The real-time monitoring method for viable E. coli O157:H7 developed in this study can be used to enrich and detect viable cells simultaneously within 24h. The unique advantages of the system developed offer great potential in the microbial analysis of food samples in routine settings.     

Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system

The development of a nanoparticle-based detection methodology for sensitive and specific DNA-based diagnostic applications is described. The technology utilizes gold nanoparticles derivatized with thiol modified oligonucleotides that are designed to bind complementary DNA targets. A glass surface with arrays of immobilized oligonucleotide capture sequences is used to capture DNA targets, which are then detected via hybridization to the gold nanoparticle probes. Amplification with silver allows for detection and quantitation by measuring evanescent wave induced light scatter with low-cost optical detection systems. Compared to Cy3-based fluorescence, silver amplified gold nanoparticle probes provide for a approximately 1000-fold increase in sensitivity. Furthermore, direct detection of non-amplified genomic DNA from infectious agents is afforded through increased specificity and even identification of single nucleotide polymorphisms (SNP) in human genomic DNA appears feasible.     

Controlled Fabrication of Gold Nanoparticle and Fluorescent Protein Conjugates

The unique optical properties of gold nanoparticles make them attractive for a wide range of applications which require optical detection and manipulation techniques. Here, we experimentally demonstrate the use of single femtosecond pulses at resonance wavelength for a controlled conjugation of gold nanoparticles and fluorescent proteins. This optically driven reaction is rigorously studied and analyzed using a variety of experimental techniques, and a detailed model is proposed which describes the adsorption of the proteins onto the nanoparticles' surface, as well as their subsequent desorption by a reducing agent. Potential applications of the resulting nanoparticle?Cprotein conjugates include controlled delivery of fluorescent markers and local sensing of biochemical processes.     

On‐target and nanoparticle‐facilitated selective enrichment of peptides and proteins for analysis by MALDI‐MS

Over the course of the last decade, a number of investigators have come to appreciate that the surface of a MALDI target, after suitable modification, can be used for selective enrichment of peptides and proteins. More recently, surface‐modified nanoparticles (NPs) that readily co‐crystallize in MALDI matrix, are not ionized by laser desorption/ionization, and do not interfere with MS have attracted interest as alternatives to surface‐modified targets for selective enrichment of peptides and proteins. Surface‐modified targets and NPs facilitate parallel processing of samples, and when used in conjunction with MALDI mass spectrometers with kHz lasers enable development of high‐throughput proteomics platforms. Targets and NPs for reversed phase and ion exchange retention, selective enrichment of glycopeptides, selective enrichment of phosphopeptides, and immunoaffinity MS are described in conjunction with details regarding their preparation and utility. Commercial availability of the reagents and substrates required to prepare surface‐modified targets and NPs is also discussed.     

A new amplification strategy for ultrasensitive electrochemical aptasensor with network-like thiocyanuric acid/gold nanoparticles

An ultrasensitive and highly specific electrochemical aptasensor for detection of thrombin based on gold nanoparticles and thiocyanuric acid is presented. For this proposed aptasensor, aptamerI was immobilized on the magnetic nanoparticles, aptamerII was labeled with gold nanoparticles. The magnetic nanoparticle was used for separation and collection, and gold nanoparticle offered excellent electrochemical signal transduction. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle/thrombin/gold nanoparticle was fabricated, and the signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles. A significant sensitivity enhancement had been obtained, and the detection limit was down to 7.82 aM. The presence of other proteins such as BSA and lysozyme did not affect the detection of thrombin, which indicates a high specificity of thrombin detection could be achieved. This electrochemical aptasensor is expected to have wide applications in protein monitoring and disease diagnosis.     

Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries

Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of "clickable" DARPins at about 30 mg per liter E. coli culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained in vitro. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications.     

Microfluidic protein detection through genetically engineered bacterial cells

Protein microarray technology, in which a large number of capture ligands are spatially arrayed at a high density, presents an attractive method for high-throughput proteomic analysis. Toward this end, we demonstrate the first cell-based protein detection in a microsystem, wherein Escherichia coli cells are genetically engineered to express the desired capture proteins on the membrane surface and are spatially arrayed as sensing elements in a microfluidic device. An E. coli clone expressing peptide ligands with high affinity and high specificity for target molecules was isolated a priori. Then these cells were electrokinetically immobilized on gold electrodes using dielectrophoresis, thus allowing each sensor element to be electrically addressable. Flow cytometry and subsequent fluorescence analysis verified the highly specific capture and detection of target molecules by the bacteria. Finally, through the coexpression of peptide-based capture ligands on the cell surface and fluorescent protein in the cytoplasm, we demonstrate an effective means of directly linking the fluorescence intensity to the density of capture ligands.