Affinity Reagents Research Articles

Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1.

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149–811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

Sequestration and Removal of Multiple Small-Molecule Contaminants Using an Optimized Aptamer-Based Ultrafiltration System.

Small-molecule toxins pose a significant threat to human health and the environment, and their removal is made challenging by their low molecular weight. Aptamers show promise as affinity reagents for binding these toxins, and recently, aptamers have been utilized for both sensing and remediation applications. We found that functionalization of ultrafiltration membranes with aptamers provides a convenient scaffold for toxin sequestration, but our initial efforts in this area were limited by low functionalization efficiencies and the ability to only capture a single target molecule. Herein, we describe detailed optimization of our aptamer-functionalized ultrafiltration membrane system and subsequent use for simultaneous removal of multiple small-molecule toxins. We examine multiple critical components involved in fabricating and functionalizing the membranes, including PEG polymer molecular weight for membrane fabrication, grafting conditions for pMAA attachment, and coupling reagents for aptamer functionalization. This screening enabled us to identify a set of unique conditions in which we were able to achieve high flux, near quantitative yield for DNA attachment, and effective overall depletion of both toxins and bacterial cells. Furthermore, we demonstrate the attachment of multiple aptamers and subsequent parallel removal of atrazine, bisphenol A, and microcystin-LR in a complex lake water matrix. Our rigorous evaluation resulted in depletion of multiple small-molecule toxins, contaminants, and microorganisms, demonstrating the potential of aptamer-functionalized membranes as point-of-use decontamination systems.

Design of Synthetic Polymer Nanoparticles That Capture and Neutralize Target Molecules

Protein affinity reagents that specifically and strongly bind to target molecules are widely used in disease detection, diagnosis, and therapy. Although antibodies and their fragments are the gold standard in protein-protein inhibitors (PPIs), synthetic polymers such as linear polymers, dendrimers, and nanoparticles as cost-effective PPIs have attracted great attention as alternatives to antibodies. These polymers exhibit high affinity to the target by imitating natural protein-protein interactions. However, only a few in vivo applications have been reported. Here, our recent advances in the development of synthetic polymers for in vivo application are reviewed. Poly(N-isopropylacrylamide) (pNIPAm) was used as a model of synthetic affinity reagents. Incorporation of both sulfated carbohydrate and hydrophobic monomers into lightly crosslinked pNIPAm nanoparticles (NPs) captured and neutralized vascular endothelial growth factor (VEGF) and inhibited tumor growth upon intravenous injection into tumor-bearing mice. Modification of a liposome with the pNIPAm-based linear polymer increased the polymer circulation time after intravenous injection and improved the affinity for the target. The pNIPAm-based NPs delivered by oral administration captured the target small molecules and inhibited their absorption from the intestine. Our recent findings provide useful information for the design of synthetic polymers that capture target molecules in vivo.

Precision Modification of Native Antibodies.

Antibodies, particularly of the immunoglobulin G (IgG) isotype, are a group of biomolecules that are extensively used as affinity reagents for many applications in research, disease diagnostics, and therapy. Most of these applications require antibodies to be modified with specific functional moieties, including fluorophores, drugs, and proteins. Thus, a variety of methodologies have been developed for the covalent labeling of antibodies. The most common methods stably attach functional molecules to lysine or cysteine residues, which unavoidably results in heterogeneous products that cannot be further purified. In an effort to prepare homogeneous antibody conjugates, bioorthogonal handles have been site-specifically introduced via enzymatic treatment, genetic code expansion, or genetically encoded tagging, followed by functionalization using bioorthogonal conjugation reactions. The resulting homogeneous products have proven superior to their heterogeneous counterparts for both in vitro and in vivo usage. Nevertheless, additional chemical treatment or protein engineering of antibodies is required for incorporation of the bioorthogonal handles, processes that often affect antibody folding, stability, and/or production yield and cost. Accordingly, concurrent with advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in site-specifically labeling native (nonengineered) antibodies without chemical or enzymatic treatments. In this review, we highlight recent strategies for producing site-specific native antibody conjugates and provide a comprehensive summary of the merits and disadvantages of these strategies.

Abstract 19: Proteome-wide biomarker discovery using digital MosaicNeedles

Abstract Changes in transcript levels detected by transcriptome profiling have been widely used as a surrogate for proteomics. However, recent studies have shown accumulating evidence that phenotypes of diseases that show correlations with protein levels cannot be explained by transcript levels. Hence a proteome-wide biomarker search will provide valuable information for cancer research that is not achievable from a strictly genomic approach. NanoMosaic is a digital immunoassay technology that uses a sub-100 nm nanoneedle sensor to detect single molecules. Over a billion nanoneedles are fabricated on a silicon substrate, thus named MosaicNeedlesTM, and formatted into 96, 384 or 1536 isolated well configurations at standard microplate dimensions. Each nanoneedle is a label-free biosensor, that is functionalized with capture antibodies. Each individual nanoneedle shifts its scattering spectrum when an antigen binds to it. The spectrum shift is further enhanced using a chemical amplification process. At low concentrations, the binding events of molecules to the MosaicNeedlesTM follow a Poisson distribution which insure that the number of molecules can be quantitated by counting the number of nanoneedles that display a spectrum change. Imaging automation is achieved by TessieTM instrument that reads the entire MosaicNeedlesTM and analyzes the color spectrum of individual nanoneedles. High multiplexing level up to 50 analytes can be achieved by parallelizing the detection in a microarray format within each well. NanoMosaic uses a hypothesis-free approach to construct its protein biomarker discovery panel, also known as proteome-wide association study (PWAS) panel. Individual protein analytes included in the PWAS panel are evenly spaced across the exome. Therefore, a sub-group of the entire human proteome or secretome can be interrogated in a hypothesis-free way before the entire proteome or a reflexive protein sub-group is interrogated. This approach addresses the limitations of the availability of the affinity reagents (antibodies, nanobodies, aptamers and etc.) that would be needed to simultaneously interrogate the whole proteome. Once valuable targets of interest are identified by running the PWAS-panel against a cohort of samples, mechanism studies can then be done using pathway specific panels whose design can be informed by previous PWAS results. In conclusion, NanoMosaic provides a solution to conduct a proteome-wide search for disease relevant biomarkers using its hypothesis-free PWAS-panels. The use of MosaicNeedlesTM provides higher sensitivity, wider dynamic range, lower cost and higher throughput than is currently possible by mass spectrometry or traditional immunoassay methods. Citation Format: Qimin Quan, Joshua Ritchey, Joe Wilkinson, John Geanacopoulos, Alaina Kaiser, John Boyce. Proteome-wide biomarker discovery using digital MosaicNeedles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 19.

A Silica Coating Approach to Enhance Bioconjugation on Metal-Encoded Polystyrene Microbeads for Bead-Based Assays in Mass Cytometry.

Bead-based assays in flow cytometry are multiplexed analytical techniques that allow rapid and simultaneous detection and quantification of a large number of analytes from small volumes of samples. The development of corresponding bead-based assays in mass cytometry (MC) is highly desirable since it could increase the number of analytes detected in a single assay. The microbeads for these assays have to be labeled with metal isotopes for MC detection. One must also be able to functionalize the bead surface with affinity reagents to capture the analytes. Metal-encoded polystyrene microbeads prepared by multi-stage dispersion polymerization can produce effective isotopic signals in MC with relatively small bead-to-bead variations. However, functionalizing this microbead surface with bioaffinity agents remains challenging, possibly due to the interference of the steric-stabilizing PVP corona on the microbead surface. Here, we report a systematic investigation of a silica coating approach to coat Eu-encoded microbeads with thin silica shells, to functionalize the surface with amino groups, and to introduce bioaffinity agents. We examine the effect of silica shell roughness on the bioconjugation capacity and the effect of silica shell thickness on signal quality in MC measurements. To limit non-specific binding, we converted the amino groups on the microbead surface to carboxylic acid groups. Antibodies were effectively attached to microbead by first conjugating NeutrAvidin to the carboxyl-modified bead surface and then attaching biotinylated antibodies to the NeutrAvidin-modified bead surface. The antibody-modified microbeads can specifically capture antigens, which were marked with isotopic labels, and generate strong signals in MC. These are promising results for the development of bead-based assays in MC.

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein Using mRNA Display.

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality, and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein, and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (KD) in the nanomolar range (15–550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect the spike protein in solution by ELISA, and the cocrystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a β-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD, and the ligands discovered in this work may find future use as reagents for diagnostic applications.

Abiotic Stimuli-Responsive Protein Affinity Reagent for IgG.

We describe an approach for the discovery of protein affinity reagents (PARs). Abiotic synthetic hydrogel copolymers can be "tuned" for selective protein capture by the type and ratios of functional monomers included in their polymerization and by the polymerization conditions (i.e., pH). By screening libraries of hydrogel nanoparticles (NPs) containing charged and hydrophobic groups against a protein target (IgG), a stimuli-responsive PAR is selected. The robust carbon backbone synthetic copolymer is rapidly synthesized in the chemistry laboratory from readily available monomers. The production of the PAR does not require living cells and is free from biological contamination. The capture and release of the protein by the copolymer NP is reversible. IgG is sequestered from human serum at pH 6.5 and following a wash step, the purified protein is released by elevating the pH to 7.3. The binding and release of the protein occur without denaturation. The abiotic material functions as a selective PAR for the F(ab')2 domain of IgG for pull-down and immunoprecipitation experiments and for isolation and purification of proteins from complex biological mixtures.

Design of synthetic polymer nanoparticles that inhibit glucose absorption from the intestine

Synthetic polymers prepared using several functional monomers have attracted attention as cost-effective protein affinity reagents and alternative to antibodies. We previously reported the synthesis of poly NIPAm-based nanoparticles (NPs) using several functional monomers that can capture target molecules. In this study, we designed NPs for capturing glucose and inhibiting intestinal absorption in living mice. For capturing glucose, we focused on the Maillard reaction between primary amines and aldehyde residues. We hypothesized that the primary amine-containing NPs can capture the open-chain structure of glucose via the Maillard reaction and inhibit intestinal absorption. NPs were prepared by the precipitation polymerization of NIPAm, N-tert-butylacrylamide (TBAm), trifluoroacetate-protected N-(3-aminopropyl)methacrylamide (T-APM), and N,N′-methylenebisacrylamide. Then, T-APM in NPs was deprotected by NH3 (aq). The amount of glucose captured by NPs depended on the percentage of TBAm and APM in vitro. After 24 h, only 2% of orally administered NPs remained in the body after administration, suggesting that many NPs were excreted without being absorbed. The prepared NPs significantly inhibited an increase in blood glucose concentration after the oral administration of glucose and NPs, indicating that NPs capture glucose and inhibit intestinal absorption. These results show the potential of using synthetic polymer nanoparticles for inhibiting postprandial hyperglycemia.

Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance.

During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP—KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3—to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3–25 nM) exhibited the lowest limits of detection (ca. μg mL−1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual’s immune response can impact the interpretation of assay results.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03317-4.

Advances in Molecularly Imprinted Polymers Based Affinity Sensors (Review).

Recent challenges in biomedical diagnostics show that the development of rapid affinity sensors is very important issue. Therefore, in this review we are aiming to outline the most important directions of affinity sensors where polymer-based semiconducting materials are applied. Progress in formation and development of such materials is overviewed and discussed. Some applicability aspects of conducting polymers in the design of affinity sensors are presented. The main attention is focused on bioanalytical application of conducting polymers such as polypyrrole, polyaniline, polythiophene and poly(3,4-ethylenedioxythiophene) ortho-phenylenediamine. In addition, some other polymers and inorganic materials that are suitable for molecular imprinting technology are also overviewed. Polymerization techniques, which are the most suitable for the development of composite structures suitable for affinity sensors are presented. Analytical signal transduction methods applied in affinity sensors based on polymer-based semiconducting materials are discussed. In this review the most attention is focused on the development and application of molecularly imprinted polymer-based structures, which can replace antibodies, receptors, and many others expensive affinity reagents. The applicability of electrochromic polymers in affinity sensor design is envisaged. Sufficient biocompatibility of some conducting polymers enables to apply them as “stealth coatings” in the future implantable affinity-sensors. Some new perspectives and trends in analytical application of polymer-based semiconducting materials are highlighted.

Accurate detection of Newcastle disease virus using proximity-dependent DNA aptamer ligation assays.

Detecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid‐phase formats are widely used for high‐performance protein detection in medical research. However, the affinity reagents used, which are mainly poly‐ and monoclonal antibodies, play an important role in the performance of PLAs. Here, we have established the first homogeneous and solid‐phase proximity‐dependent DNA aptamer ligation assays for rapid and accurate detection of Newcastle disease virus (NDV). NDV is detected by a pair of extended DNA aptamers that, upon binding in proximity to proteins on the envelope of the virus, are joined by enzymatic ligation to form a unique amplicon that can be sensitively detected using real‐time PCR. The sensitivity, specificity, and reproducibility of the assays were validated using 40 farm samples. The results demonstrated that the developed homogeneous and solid‐phase PLAs, which use NDV‐selective DNA aptamers, are more sensitive than the sandwich enzymatic‐linked aptamer assay (ELAA), and have a comparable sensitivity to real‐time reverse transcription PCR (rRT‐PCR) as the gold standard detection method. In addition, the solid‐phase PLA was shown to have a greater dynamic range with improved lower limit of detection, upper‐ and lower limit of quantification, and minimal detectable dose as compared with those of ELAA and rRT‐PCR. The specificity of PLA is shown to be concordant with rRT‐PCR.

Immunogenicity Risk Profile of Nanobodies.

Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or in vivo imaging reagents entering the clinic. They possess unique characteristics, including a minimal size, providing fast pharmacokinetics, high-target specificity, and an affinity in the (sub-)nanomolar range in conjunction with an easy selection and production, which allow them to outperform conventional antibodies for imaging and radiotherapeutic purposes. As for all protein theranostics, extended safety assessment and investigation of their possible immunogenicity in particular are required. In this study, we assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) for PET imaging of tumor-associated macrophages. For the anti-HER2 Nb, we show that only one out of 20 patients had a low amount of pre-existing anti-drug antibodies (ADAs), which only marginally increased 3 months after administering the Nb, and without negative effects of safety and pharmacokinetics. Further in vitro immunogenicity assessment assays showed that both non-humanized Nbs were taken up by human dendritic cells but exhibited no or only a marginal capacity to activate dendritic cells or to induce T cell proliferation. From our data, we conclude that monomeric Nbs present a low immunogenicity risk profile, which is encouraging for their future development toward potential clinical applications.One Sentence SummaryNanobodies, the recombinant single domain affinity reagents derived from heavy chain-only antibodies in camelids, are proven to possess a low immunogenicity risk profile, which will facilitate a growing number of Nanobodies to enter the clinic for therapeutic or in vivo diagnostic applications.

Synthesis of a High Affinity Complementary Peptide-Polymer Nanoparticle (NP) Pair Using Phage Display.

Peptide-polymer complementary pairs can provide useful tools for isolating, organizing, and separating biomacromolecules. We describe a procedure for selecting a high affinity complementary peptide-polymer nanoparticle (NP) pair using phage display. A hydrogel copolymer nanoparticle containing a statistical distribution of negatively charged and hydrophobic groups was used to select a peptide sequence from a phage displayed library of >1010 peptides. The NP has low nanomolar affinity for the selected cyclic peptide and exhibited low affinity for a panel of diverse proteins and peptide variants. Affinity arises from the complementary physiochemical properties of both NP and peptide as well as the specific peptide sequence. Comparison of linear and cyclic variants of the peptide established that peptide structure also contributes to affinity. These findings offer a general method for identifying polymer-peptide complementary pairs. Significantly, precise polymer sequences (proteins) are not a requirement, a low information statistical copolymer can be used to select for a specific peptide sequence with affinity and selectivity comparable to that of an antibody. The data also provides evidence for the physiochemical and structural contributions to binding. The results confirm the utility of abiotic, statistical, synthetic copolymers as selective, high affinity peptide affinity reagents.

Redesigning the Genetic Polymers of Life.

Genomes can be viewed as constantly updated memory systems where information propagated in cells is refined over time by natural selection. This process, commonly known as heredity and evolution, has been the sole domain of DNA since the origin of prokaryotes. Now, some 3.5 billion years later, the pendulum of discovery has swung in a new direction, with carefully trained practitioners enabling the replication and evolution of "xeno-nucleic acids" or "XNAs"-synthetic genetic polymers in which the natural sugar found in DNA and RNA has been replaced with a different type of sugar moiety. XNAs have attracted significant attention as new polymers for synthetic biology, biotechnology, and medicine because of their unique physicochemical properties that may include increased biological stability, enhanced chemical stability, altered helical geometry, or even elevated thermodynamics of Watson-Crick base pairing.This Account describes our contribution to the field of synthetic biology, where chemical synthesis and polymerase engineering have allowed my lab and others to extend the concepts of heredity and evolution to synthetic genetic polymers with backbone structures that are distinct from those found in nature. I will begin with a discussion of α-l-threofuranosyl nucleic acid (TNA), a specific type of XNA that was chosen as a model system to represent any XNA system. I will then proceed to discuss advances in organic chemistry that were made to enable the synthesis of gram quantities of TNA phosphoramidites and nucleoside triphosphates, the monomers used for solid-phase and polymerase-mediated TNA synthesis, respectively. Next, I will recount our development of droplet-based optical sorting (DrOPS), a single-cell microfluidic technique that was established to evolve XNA polymerases in the laboratory. This section will conclude with structural insights that have been gained by solving X-ray crystal structures of a laboratory-evolved TNA polymerase and a natural DNA polymerase that functions with general reverse transcriptase activity on XNA templates.The final passage of this Account will examine the role that XNAs have played in synthetic biology by highlighting examples in which engineered polymerases have enabled the evolution of biologically stable affinity reagents (aptamers) and catalysts (XNAzymes) as well as the storage and retrieval of binary information encoded in electronic word and picture file formats. Because these examples provide only a glimpse of what the future may have in store for XNA, I will conclude the Account with my thoughts on how synthetic genetic polymers could help drive new innovations in synthetic biology and molecular medicine.

Isolation of Natural DNA Aptamers for Challenging Small-Molecule Targets, Cannabinoids.

Aptamers are nucleic acid-based affinity reagents that are isolated via an in vitro process known as systematic evolution of ligands by exponential enrichment (SELEX). Despite their great potential for a wide range of analytical applications, there are relatively few high-quality small-molecule binding aptamers, especially for "challenging" targets that have low water solubility and/or limited moieties for aptamer recognition. The use of libraries containing chemically modified bases may improve the outcome of some SELEX experiments, but this approach is costly and yields inconsistent results. Here, we demonstrate that a thoughtfully designed SELEX procedure with natural DNA libraries can isolate aptamers with high affinity and specificity for challenging small molecules, including targets for which such selections have previously failed. We first isolate a DNA aptamer with nanomolar affinity and high specificity for (-)-trans-Δ9-tetrahydrocannabinol (THC), a target previously thought to be unsuitable for SELEX with natural DNA libraries. We subsequently isolate aptamers that exhibit high affinity and cross-reactivity to two other challenging targets, synthetic cannabinoids UR-144 and XLR-11, while maintaining excellent specificity against a wide range of non-target interferents. Our findings demonstrate that natural nucleic acid libraries can yield high-quality aptamers for small-molecule targets, and we outline a robust workflow for isolating other such aptamers in future selection efforts.

Fully Integrated and Multiplexed Sample Preparation Technology for Sensitive Interactome Profiling.

Affinity purification coupled to mass spectrometry (AP-MS) is a popular approach for deciphering the architecture of protein interaction networks. Protein lysates (100 μg) are typically required for multistep sample processing in large volumes, which often causes sample loss and reduces the MS analysis sensitivity. Herein, we reported a fully integrated spintip-based AP-MS technology, termed FISAP, for multiplexed and sensitive interactome profiling. The FISAP device can be easily employed for routine use by introducing AP beads into a C18 StageTip. Taking advantage of the switchable functionalization of the C18 matrix by sodium dodecyl sulfate, all the sample preparation steps encompassing peptide or antibody-based AP, reduction, alkylation, tryptic digestion, tandem mass tag (TMT) labeling, and desalting can be performed in a single tip with a benchtop centrifuge in 4 h. Using a biotinylated tyrosine phosphorylated (pTyr) peptide as an affinity ligand, we mapped the pTyr-dependent interactome of the pY191 motif on the immune receptor CD28 cytoplasmic domain. When processing 50 μg of protein lysates, FISAP showed a comparable interactome identification performance but better quantification performance and lower background interference compared to the traditional tube-based method. Furthermore, a cost-effective on-column TMT labeling protocol was established and integrated into the FISAP pipeline with increased sensitivity. Compared to the tube-based method, the usage of a synthetic peptide probe and a TMT reagent was both reduced by 20 times. As low as 1 μg of protein lysates could be applied for interactome profiling. Finally, we expanded the applicability of the FISAP technology to epitope tag-based AP-MS for profiling the ILK/PINCH/Parvin complex using 100 times less protein lysate than a previous report. Collectively, FISAP is an easy-to-use and sensitive technology for quantitatively profiling protein complexes when the starting material and affinity reagent are the limitation, especially for applications in biomedical research and chemical biology.

Direct Digital Sensing of Proteins in Solution through Single-Molecule Optofluidics

Highly sensitive detection of proteins is of central importance to biomolecular analysis and diagnostics. Conventional protein sensing assays, such as ELISAs, remain reliant on surface-immobilization of target molecules and multi-step washing protocols for the removal of unbound affinity reagents. These features constrain parameter space in assay design, resulting in fundamental limitations due to the underlying thermodynamics and kinetics of the immunoprobe-analyte interaction. Here, we present a new experimental approach for the quantitation of protein analytes through the implementation of an immunosensor assay that operates fully in solution and realises rapid removal of excess probe prior to detection without the need of washing steps.

Affinity-based proteomics reveals novel targets of inositol pyrophosphate (5-IP7 )-dependent phosphorylation and binding in Trypanosoma cruzi replicative stages.

Diphosphoinositol-5-pentakisphosphate (5-PP-IP5 ), also known as inositol heptakisphosphate (5-IP7 ), has been described as a high-energy phosphate metabolite that participates in the regulation of multiple cellular processes through protein binding or serine pyrophosphorylation, a posttranslational modification involving a β-phosphoryl transfer. In this study, utilizing an immobilized 5-IP7 affinity reagent, we performed pull-down experiments coupled with mass spectrometry identification, and bioinformatic analysis, to reveal 5-IP7 -regulated processes in the two proliferative stages of the unicellular parasite Trypanosoma cruzi. Our protein screen clearly defined two cohorts of putative targets either in the presence of magnesium ions or in metal-free conditions. We endogenously tagged four protein candidates and immunopurified them to assess whether 5-IP7 -driven phosphorylation is conserved in T. cruzi. Among the most interesting targets, we identified a choline/o-acetyltransferase domain-containing phosphoprotein that undergoes 5-IP7 -mediated phosphorylation events at a polyserine tract (Ser578-580 ). We also identified a novel SPX domain-containing phosphoribosyltransferase [EC 2.7.6.1] herein termed as TcPRPPS4. Our data revealed new possible functional roles of 5-IP7 in this divergent eukaryote, and provided potential new targets for chemotherapy.

Selection and characterisation of Affimers specific for CEA recognition

Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.