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In this study, the affinity potentials of biosynthesized and functionalized iron oxide nanoparticles (IONPs), gold nanoparticles (AuNPs), and iron-gold nanocomposites (Fe3O4-AuNCs) harnessed to target enteric pathogens were evaluated. Colloidal IONPs and AuNPs biosynthesized by Bacillus subtilis were monitored and characterized with UV–Vis spectroscopy, Fourier transform infrared (FTIR), scanning electron microscope (SEM), energy dispersive X-ray (EDX), and X-ray diffractometer (XRD). The synthesized nanoparticles were functionalized independently and as nanocomposites with the ligand (ethylenediaminetetraacetic acid (EDTA)) and polymers (polyethyleneimine (PEI) and polyethylene glycol (PEG)). Affinity capture of the derived functionalized nanoparticles and nanocomposites was evaluated on enteric bacteria and monitored at 540–600 nm wavelengths. Colloidal suspensions of the biosynthesized IONPs and AuNPs showed UV–Vis absorption peaks at 261 and 548 nm and mean distribution sizes of 72 and 83 nm, respectively. The energy dispersive X-ray (EDX) analysis showed 48.59% zerovalent iron oxide and 51.26% zerovalent gold. X-ray diffractometer (XRD) reported Bragg's peaks corresponding with the crystal lattice of iron oxide and gold nanoparticles. Capped Fe3O4-AuNCs functionalized with PEI had a significantly higher ratio of zerovalent gold (75.56%) compared to capped Fe3O4-AuNCs functionalized with PEG (61.40%); however, an increased zerovalent iron oxide (20.00%) than PEI-functionalized Fe3O4-AuNCs (7.40%). Capped Fe3O4-AuNCs showed significant binding affinities with Bacillus cereus, Salmonella typhi, Staphylococcus aureus, and Klebsiella aerogenes. The results show that ligands and polymers could enhance Fe3O4-AuNCs' ability to interact and bind to enteric bacterial pathogen's negatively charged cell wall (teichoic acid) and outer membrane (lipopolysaccharides). Biosynthesized and functionalized IONPs, AuNPs, and Fe3O4-AuNCs provide cost-effective and feasible nano-based detection of enteric bacteria. [ABSTRACT FROM AUTHOR]

Antibodies for treatment and prophylaxis against SARS-CoV-2 are needed particularly for immunocompromised individuals, who cannot adequately benefit from vaccination. To address this need, Aerium Therapeutics is developing antibodies targeting the SARS-CoV-2 spike protein. A bioanalytical method to quantify fully human monoclonal antibodies in a population with widely varying anti-spike antibody titers is required to investigate the pharmacokinetics of these antibodies in clinical trials. To eliminate interference from endogenous anti-spike protein antibodies, an HPLC–MS/MS assay was developed to quantify the investigational monoclonal antibodies (AER001 and AER002) by targeting signature peptides spanning the monoclonal antibodies' CDR regions. By optimizing and comparing affinity capture and ammonium sulphate precipitation, it was demonstrated that both procedures allowed accurate and precise quantification of AER001 and AER002 in human serum with comparable sensitivity. Ammonium sulphate precipitation outperformed immunocapture due to its simplicity and speed at lower cost and a full bioanalytical method validation was performed in human serum. The assay was also validated for human nasal lining fluid extract with a 50-fold lower limit of quantification and was shown to deliver similar sensitivity to previously published affinity capture HPLC–MS/MS assays. Finally, the CDR-derived signature peptides were also generated by tryptic digestion of blank serum in some individuals, an important caveat for HPLC–MS/MS strategies targeting human monoclonal antibodies. In summary, the presented results show that ammonium sulphate precipitation and HPLC–MS/MS allow accurate and precise quantification of monoclonals in clinical studies. The developed methods demonstrate that HPLC–MS/MS can reliably quantify human monoclonal antibodies even when endogenous antibodies with overlapping specificities are present and are crucial for the clinical testing of two investigational COVID-19 monoclonals. [ABSTRACT FROM AUTHOR]

A milestone in the field of recombinant binding molecules was achieved 30 years ago with the discovery of single-domain antibodies from which antigen-binding variable domains, better known as nanobodies (Nbs), can be derived. Being only one tenth the size of conventional antibodies, Nbs feature high affinity and specificity, while being highly stable and soluble. In addition, they display accessibility to cryptic sites, low off-target accumulation and deep tissue penetration. Efficient selection methods, such as (semi-)synthetic/naïve or immunized cDNA libraries and display technologies, have facilitated the isolation of Nbs against diverse targets, and their single-gene format enables easy functionalization and high-yield production. This Review highlights recent advances in Nb applications in various areas of biological research, including structural biology, proteomics and high-resolution and in vivo imaging. In addition, we provide insights into intracellular applications of Nbs, such as live-cell imaging, biosensors and targeted protein degradation. [ABSTRACT FROM AUTHOR]

We report the development of a new electron-rich aniline (ERA)-based cleavable linker. Anilines can be incorporated into peptides during SPPS and are stable to most reaction conditions. ERA-containing peptides can be cleaved rapidly in the presence of oxidants, such as DDQ, CAN, and NaIO 4 , in 30 min at room temperature. The compatibility of these conditions is demonstrated on peptides containing natural and unnatural amino acids. While the cleavages of other oxidatively labile linkers is known to cause decomposition of Tyr and Trp, these sensitive residues are stable to DDQ oxidations. An ERA-linker also enables the capture and release of a biotinylated peptide from immobilized streptavidin. ERA-linkers may serve as an excellent tool for peptide library screening applications., Competing Interests: Declaration of competing interest The authors declare no financial interests or personal relationships that are potential competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The combination of advanced mass spectrometry and enrichment-based sample preparation methods has enhanced analytical capabilities in clinical proteomics. In this chapter, we describe a method of proteome analysis to identify Borrelia-derived peptides in urine that includes a sample affinity enrichment method coupled with liquid chromatography tandem mass spectrometry analysis and a bioinformatic peptide authentication algorithm., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mango allergy is a rare condition, which may cause severe hypersensitivity reactions, such as anaphylaxis, angioedema, asthma and contact dermatitis. By exploiting the combinatorial peptide ligand library (CPLL) technology, mango proteomes have been extracted and the presence of traces of allergens assessed via Western blot analysis two-dimensional maps. Upon reactive spot elution and mass spectrometry analyses, four major mango allergens could be identified for the first time and shown to be in common with three of the five known banana species. These allergens include: Mus a 1, Mus a 2 and Mus a 5. Additional mango allergens detected do not seem to be in common with the banana species. In particular, a pectinesterase and a superoxide dismutase, both widely described as allergens, could be identified in mango extracts. Conversely, plain mango extracts not treated with CPLLs did not exhibit any reactive spots in Western blot analysis. [ABSTRACT FROM AUTHOR]

A novel method for rapid screening of antimicrobial peptides (AMPs) was developed by using immobilized lipid affinity capture (ILAC) coupled with LC-MS. Phospholipid (PL) mixture containing phosphatidyl glycerol (PG): phosphatidyl ethanolamine (PE) (4:1), roughly mimic the PL composition of Gram-positive bacterial membrane, was covalently immobilized on magnetic particles (MPs). PL monolayer immobilized on MPs was used as a matrix for capturing of the membrane-disruptive AMPs. Hominicin, a new AMP against Gram-positive bacteria, was successfully captured by ILAC from the peptide pool of Staphylococcus hominis MBBL 2-9. The hominicin was identified by the comparative analysis of LC-MS 2Dprofiles of peptides captured by bare and PL-immobilized MPs. This is the first report for the development of rapid AMP screening method using lipid-immobilized MPs and LC-MS which will be a promising tool for discovery of various kinds of AMPs. [ABSTRACT FROM AUTHOR]

Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 μg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method.ᅟ [ABSTRACT FROM AUTHOR]

Introduction: Mass spectrometry (MS) is the premier tool for discovering novel disease-associated protein biomarkers. Unfortunately, when applied to complex body fluid samples, MS has poor sensitivity for the detection of low abundance biomarkers (≪10 ng/mL), derived directly from the diseased tissue cells or pathogens. Areas covered: Herein we discuss the strengths and drawbacks of technologies used to concentrate low abundance analytes in body fluids, with the aim to improve the effective sensitivity for MS discovery. Solvent removal by dry-down or dialysis, and immune-depletion of high abundance serum or plasma proteins, is shown to have disadvantages compared to positive selection of the candidate biomarkers by affinity enrichment. A theoretical analysis of affinity enrichment reveals that the yield for low abundance biomarkers is a direct function of the binding affinity (Association/Dissociation rates) used for biomarker capture. In addition, a high affinity capture pre processing step can effectively dissociate the candidate biomarker from partitioning with high abundance proteins such as albumin. Expert commentary: Properly designed high affinity capture materials can enrich the yield of low abundance (0.1-10 picograms/mL) candidate biomarkers for MS detection. Affinity capture and concentration, as an upfront step in sample preparation for MS, combined with MS advances in software and hardware that improve the resolution of the chromatographic separation can yield a transformative new class of low abundance biomarkers predicting disease risk or disease latency. [ABSTRACT FROM AUTHOR]

The attachment of biotin to a small molecule provides a powerful tool in biology. Here, we present a systematic approach to identify biotinylated analogues of the Hsp90 inhibitor PU-H71 that are capable of permeating cell membranes so as to enable the investigation of Hsp90 complexes in live cells. The identified derivative 2g can isolate Hsp90 through affinity purification and, as we show, represents a unique and useful tool to probe tumor Hsp90 biology in live cells by affinity capture, flow cytometry and confocal microscopy. To our knowledge, 2g is the only reported biotinylated Hsp90 probe to have such combined characteristics.

The pharmaceutical industry has been continually challenged by dwindling target diversity. To obviate this trend, phenotypic screens have been adopted, complementing target-centric screening approaches. Phenotypic screens identify drug leads using clinically relevant and translatable mechanisms, remaining agnostic to targets. While target anonymity is advantageous early in the drug discovery process, it poses challenges to hit progression, including the development of backup series, retaining desired pharmacology during optimization, discovery of markers, and understanding mechanism-driven toxicity. Consequently, significant effort has been expended to elaborate the targets and mechanisms at work for promising screening hits. Affinity capture is commonly leveraged, where the compounds are linked to beads and targets are abstracted from cell homogenates. This technique has proven effective for identifying targets of kinase, PARP, and HDAC inhibitors, and examples of new targets have been reported. Herein, a three-pronged approach to target deconvolution by affinity capture is described, including the implementation of a uniqueness index that helps discriminate between bona fide targets and background. The effectiveness of this approach is demonstrated using characterized compounds that act on known and noncanonical target classes. The platform is subsequently applied to phenotypic screening hits, identifying candidate targets. The success rate of bead-based affinity capture is discussed. [ABSTRACT FROM AUTHOR]

Novel mass spectrometric immunoassays (MSIAs) for the isolation and structural characterization of plasma apolipoprotein A-I (apoA-I), apoA-II, and apoE have been developed. The assays combine selective isolation of apolipoprotein species via affinity capture with mass-specific detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In application, plasma (from 50 μl of whole blood drawn from individuals, using finger lancet) was addressed with affinity pipette tips derivatized with antibodies toward the specific apolipoprotein. The time required for each assay was ∼15 min, less if assays on multiple individuals were performed in parallel. In a brief study of five individuals, several recently reported apoA-II variants were identified and observed consistently in all individuals. Additionally, the apoE phenotype of E3/E3 was observed in three of the individuals, and E2/E3 and E3/E4 observed in the remaining two individuals, the latter of whom suffers from Alzheimer’s disease.Overall, the MSIA approach offers a rapid, sensitive, and highly accurate means of profiling apolipoproteins from small volumes of plasma.

During their proliferation and the host's concomitant attempts to suppress it, LINE-1 (L1) retrotransposons give rise to a collection of heterogeneous ribonucleoproteins (RNPs); their protein and RNA compositions remain poorly defined. The constituents of L1-associated macromolecules can differ depending on numerous factors, including, for example, position within the L1 life cycle, whether the macromolecule is productive or under suppression, and the cell type within which the proliferation is occurring. This chapter describes techniques that aid the capture and characterization of protein and RNA components of L1 macromolecules from tissues that natively express them. The protocols described have been applied to embryonal carcinoma cell lines that are popular model systems for L1 molecular biology (e.g., N2102Ep, NTERA-2, and PA-1 cells), as well as colorectal cancer tissues. N2102Ep cells are given as the use case for this chapter; the protocols should be applicable to essentially any tissue exhibiting endogenous L1 expression with minor modifications., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: The properties of a transducer's surface are crucial in biosensor development since they strongly condition the chemical routes applicable for bioreceptor incorporation and subsequent surface physical blocking. In this way, surface (bio)engineering should provide specificity against the target of choice and minimal non-specific adsorption of non-target components onto the active area of the sensor. The objective of this work was to study the effect of different surface treatments on SPR sensor functionalization and optical performance. We used as a model the well-known thrombin-binding aptamer (TBA), which was incorporated on surface via two different paths: direct selfassembly of thiolated TBA and affinity capture of biotinylated TBA by physisorbed neutravidin. Our results indicate that chip pre-treatment with oxygen plasma produced better transducers than pre-treatment by immersion in organic solvents, piranha or KOH solution, independently of the biofunctionalization strategy used for aptasensor production. [Copyright &y& Elsevier]

We describe a fluorogenic assay for activated protein C (APC) by using magnetic beads modified with DNA aptamers, taking advantage of strong binding affinity of aptamer, facile magnetic separation, and signal amplification via an enzymatic reaction. APC is specifically captured from a sample by the DNA aptamers on magnetic beads, and the concentrated APC then catalyzes the conversion of a fluorogenic substrate of APC to a fluorescent product. Detection of APC is achieved by measuring the generated product. This method is simple, sensitive, and specific. APC can be detected at 0.4 pM concentration level in a sample volume of 250 μL, corresponding to 0.1 femtomole of APC, when 2-h enzymatic reaction is employed. The proteins thrombin, trypsin, proteinase K, chymotrypsin, and elastase do not interfere. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Development of micro-well array systems for use in high-throughput screening of rare cells requires a detailed understanding of the factors that impact the specific capture of cells in wells and the distribution statistics of the number of cells deposited into wells. In this study we investigate the development of microbubble (MB) well array technology for sorting antigen-specific B-cells. Using Poisson statistics we delineate the important role that the fractional area of MB well opening and the cell seeding density have on determining cell seeding distribution in wells. The unique architecture of the MB well hinders captured cells from escaping the well and provides a unique microenvironmental niche that enables media changes as needed for extended cell culture. Using cell lines and primary B and T cells isolated from human peripheral blood we demonstrate the use of affinity capture agents coated in the MB wells to enrich for the selective capture of B cells. Important differences were noted in the efficacy of bovine serum albumin to block the nonspecific adsorption of primary cells relative to cell lines as well as the efficacy of the capture coatings using mixed primary B and T cells samples. These results emphasize the importance of using primary cells in technology development and suggest the need to utilize B cell capture agents that are insensitive to cell activation. [ABSTRACT FROM AUTHOR]

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine, and 5-propynyl-2′-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments in mixtures. [ABSTRACT FROM AUTHOR]

Traditionally, proteomics is the high-throughput characterization of the global complement of proteins in a biological system using cutting-edge technologies (robotics and mass spectrometry) and bioinformatics tools (Internet-based search engines and databases). As the field of proteomics has matured, a diverse range of strategies have evolved to answer specific problems. Chemical proteomics is one such direction that provides the means to enrich and detect less abundant proteins (the 'hidden' proteome) from complex mixtures of wide dynamic range (the 'deep' proteome). In pharmacology, chemical proteomics has been utilized to determine the specificity of drugs and their analogues, for anticipated known targets, only to discover other proteins that bind and could account for side effects observed in preclinical and clinical trials. As a consequence, chemical proteomics provides a valuable accessory in refinement of second- and third-generation drug design for treatment of many diseases. However, determining definitive affinity capture of proteins by a drug immobilized on soft gel chromatography matrices has highlighted some of the challenges that remain to be addressed. Examples of the different strategies that have emerged using well-established drugs against pharmaceutically important enzymes, such as protein kinases, metalloproteases, PDEs, cytochrome P450s, etc., indicate the potential opportunity to employ chemical proteomics as an early-stage screening approach in the identification of new targets. [ABSTRACT FROM AUTHOR]

The progressive understanding and improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), realized over the years through the considerable efforts of Dr. Marvin Vestal, have made possible numerous comparable efforts involving its application in the biological sciences. Here we revisit the concepts behind one such analytical approach, Mass Spectrometric Immunoassay, which is designed to selectively detect and quantify proteins present in biological milieu. [ABSTRACT FROM AUTHOR]

Abstract: Antibody–drug conjugates (ADCs) are designed to facilitate the targeted delivery of cytotoxic drugs to improve their tumor fighting effects and minimize systemic toxicity. However, efficacy and safety can potentially be compromised due to the release of conjugated drugs from the ADC with time while in circulation, resulting in changes in the drug-to-antibody ratio (DAR). Current understanding of this process is limited because existing methods such as immunoassays fail to distinguish ADCs with different DARs. Here we demonstrate a novel method with bead-based affinity capture and capillary liquid chromatography–mass spectrometry to allow direct measurement of drug release by quantifying DAR distributions of the ADC in plasma/serum. This method successfully identified individual intact conjugated antibody species produced due to drug loss from ADCs (e.g., an engineered site-specific anti-MUC16 THIOMAB–drug conjugate) and measured the corresponding DAR distributions in vitro and in vivo. Information obtained can provide insights into the mechanisms involved in drug loss and help to optimize ADC therapeutics. Other potential applications of the method may include characterization of posttranslational modifications, protein adducts, and immunogenicity. [Copyright &y& Elsevier]

Abstract: An ultra-sensitive sandwich ELISA was developed for detection of AFM1 in milk. The assay involved the immobilization of rat monoclonal antibody of AFM1 in 384 microtiter plate to capture AFM1 antigen. This was detected by tracer secondary rabbit poly-clonal antibody labelled with horseradish peroxidase upon addition of a luminol-based substrate. Milk samples with different fat percentage were analyzed after pre-treatment. Linear range of AFM1 detection 250–6.25pg/mL was achieved in 3% fat milk. The miniaturised assay (10μL) enabled ultra trace analysis of AFM1 in milk with much improved lower limit of detection at 0.005pg/mL. A sensitive magnetic nanoparticles (MNPs) based ELISA was also developed and coupled with micro plate ELISA for analysis in milk. The hybrid-assay, by coupling the 1°Ab immobilized MNPs column with microwell plate assay enabled simultaneous measurement of low (0.5pg/mL) and high AFM1 contamination (200pg/mL). The most promising feature of this MNPs-ELISA is the small column size, high capture efficiency and lower cost over other reported materials. The proposed assay can be deployed for simultaneous analysis and monitoring of AFM1 in milk. [ABSTRACT FROM AUTHOR]

Abstract: Cell surface receptors and their associated signaling pathways on the plasma membrane are key targets in understanding cellular responses. However, the isolation and identification of receptor complexes has been elusive. The Fc receptor was captured from the surface of live cells using microbeads coated with the receptor’s cognate ligand, gamma globulin (IgG), and analyzed by liquid chromatography and tandem mass spectrometry (LC–MS/MS) alongside several controls. Live-cell affinity receptor chromatography (LARC) resulted in a partially nonredundant list of 288 proteins that were specific to the Fc receptor complex. The proteins identified were in close agreement with previously determined factors in the Fc receptor complex as demonstrated by genetic and biochemical methods and revealed novel complex members. Confocal microscopy was used to confirm recruitment of SRC, SYK, PLC, PKC, PI3K, SHIP, TEC, CDC42, RAP, PAK, GAP, GEF, GRP, and CRK to the receptor complex upon activation by the same ligand microbeads. The expression of mutants and silencing RNA against specific isoforms were used to demonstrate a functional role for novel members of the Fc receptor complex, including RHOG (RAS homologue member G), p115 RhoGEF (protein of 115-kDa RAS homologue guanine exchange factor), and CRKL (CRK-like). The recruitment of AKT pleckstrin homology (PH) domain green fluorescent protein (GFP) was used to quantify the production of phosphorylated inositol at the activated receptor complex. We conclude that it is feasible to capture an activated receptor complex from the surface of live cells using ligand-coated microbeads for identification of members of a receptor complex or pathway by LC–MS/MS. [Copyright &y& Elsevier]

Abstract: DNA–protein binding is among the most frequently studied biomolecular interactions with high importance in modern systems biology research. One interesting aspect of this rapidly developing field is the affinity capture of proteins by G-quartet forming oligonucleotides also referred to as aptamers. G-quartets are structural motifs formed by guanine-rich sequences commonly occurring in the human genome. In this paper, we describe a capillary gel electrophoresis based method to validate G-quartet formation of in-house designed oligonucleotides and discuss the effect of monovalent cation concentration on the development of this structure. The relevant aptamer was then bound to magnetic beads to form an affinity capture surface for target proteins, which were then analyzed by matrix-assisted laser desorption/ionization mass spectrometry. [Copyright &y& Elsevier]

In vitro quantification of the catalytic activity of an enzyme isoform requires the availability of selective agents that allow for either measurements in the presence of the other enzyme isoforms or purification of the isoform and subsequent performance of these measurements on the purified enzyme. Isozyme-specific antibodies are useful tools for these types of analyses. In the present report, we detail a method for the measurement of phospholipase C-γ1 enzyme activity employing native enzyme that is immobilized on microtiter plates. The method uses biotinylated antiglobulin bound to streptavidin-coated microtiter plates to immobilize antiphospholipase C-γ1 antibody and subsequently capture phospholipase C-γ1 from brain tissue lysates. This method avoids biotinylation of the primary (antiphospholipase C-γ1) antibody, making it less labor intensive than previously described methods for using streptavidin-coated plates; in addition, it is highly reproducible and sensitive and allows for quantification of enzyme activity. We employ the technique to show that one or more tyrosine kinases copurify with rat brain phospholipase C-γ1. The method is applicable to the study of any enzyme isoform for which antibodies that capture the native form of the enzyme are available and could easily be employed in high-throughput procedures. [Copyright &y& Elsevier]

Antibody-based affinity capture has become the gold standard in sample preparation for determination of low-abundance protein biomarkers in biological matrices prior to liquid chromatography-mass spectrometry (LC-MS) determination. This comprises both capture of intact proteins prior to the digestion step and capture of proteolytic peptides after digestion of the sample. The latter can be performed both using antibodies specifically developed to capture target proteolytic peptides, as well as by the less explored use of anti-protein antibodies to capture the proteolytic epitope peptide. Molecularly imprinted polymers (MIPs), also called plastic antibodies are another affinity-based approach emerging as sample preparation technique in LC-MS based protein biomarker analysis. The current review gives a critical and comprehensive overview of proteolytic peptide capture using antibodies and MIPs in LC-MS based protein biomarker determination during the last five years. The main emphasis is on capture of non-modified peptides, while a brief overview of affinity capture of peptides containing post-translational modifications (PTMs) is provided. [Display omitted] • Protein biomarker determination using peptide affinity capture. • Antibodies and molecularly imprinted polymers for sample clean-up prior to LC-MS. • Tools for determination of low-abundance biomarkers. • Determination of post-translational modifications by immuno-MS. • Recent progress in peptide affinity capture. [ABSTRACT FROM AUTHOR]

Blood serum or plasma proteins are potentially useful in COVID-19 research as biomarkers for risk prediction, diagnosis, stratification, and treatment monitoring. However, serum protein-based biomarker identification and validation is complicated due to the wide concentration range of these proteins, which spans more than ten orders of magnitude. Here we present a combined affinity purification-liquid chromatography mass spectrometry approach which allows identification and quantitation of the most abundant serum proteins along with the nonspecifically bound and interaction proteins. This led to the reproducible identification of more than 100 proteins that were not specifically targeted by the affinity column. Many of these have already been implicated in COVID-19 disease., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Studying protein complexes in vitro requires the production of a relatively pure sample that maintains the full complement, native organization, and function of that complex. This can be particularly challenging to achieve for large, multi-component, membrane embedded complexes using the traditional recombinant expression and reconstitution methodologies. However, using affinity capture from native cells, suitable whole endogenous protein complexes can be isolated. Here we present a protocol for the affinity isolation of baker's yeast (S. cerevisiae) nuclear pore complexes, which are ~50 MDa assemblies made up of 552 distinct proteins and embedded in a double-membraned nuclear envelope. Producing this sample allowed us for the first time to perform analyses to characterize the mass, stoichiometry, morphology, and connectivity of this complex and to obtain its integrative structure with ~9 Å precision. We believe this methodology can be applied to other challenging protein complexes to produce similar results., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Both capillary electrophoresis (CE) and mass spectrometry (MS) technologies are powerful analytical tools that have been used extensively in the characterization of biologics in the biopharmaceutical industry. The direct coupling of CE with MS is an attractive approach, in that the high separation capability of CE and the ultrasensitive detection and accurate identification performance of MS can be combined to provide a powerful system for the analysis of complex analytes. In this chapter, we discuss the detailed procedure of carrying out CE-MS analysis using a nano sheath-flow interface and its applications including intact mass analysis of monoclonal antibodies and fusion proteins, and a biotransformation study of two Fc-FGF21 molecules in a single-dose pharmacokinetic mice study. Optimization processes, including the finetuning of CE conditions and MS parameters, are illustrated in this chapter, with focuses on method robustness and assay reproducibility., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Recent progress in the development of affinity grids for cryoelectron microscopy (cryo-EM) typically employs genetic engineering of the protein sample such as histidine or Spy tagging, immobilized antibody capture, or nonselective immobilization via electrostatic interactions or Schiff base formation. We report a powerful and flexible method for the affinity capture of target proteins for cryo-EM analysis that utilizes small-molecule ligands as bait for concentrating human target proteins directly onto the grid surface for single-particle reconstruction. This approach is demonstrated for human p97, captured using two different small-molecule high-affinity ligands of this AAA+ ATPase. Four electron density maps are revealed, each representing a p97 conformational state captured from solution, including a double-hexamer structure resolved to 3.6 Å. These results demonstrate that the noncovalent capture of protein targets on EM grids modified with high-affinity ligands can enable the structure elucidation of multiple configurational states of the target and potentially inform structure-based drug design campaigns.

Background: Surrogate monoclonal antibodies (mAbs) used in preclinical in vivo studies can be challenging to quantify due to lack of suitable immunoaffinity reagents or unavailability of the mAb protein sequence. Generic immunoaffinity reagents were evaluated to develop sensitive LC-MS/MS assays. Peptides of unknown sequence can be used for selective LC-MS quantification. Results: anti-mouse IgG1 was found to be an effective immunoaffinity reagent, enabling quantification of mouse IgG1 mAbs in mouse serum. Selective peptides of unknown sequence were applied for multiplex LC-MS quantification of two rat mAbs co-dosed in mouse. Conclusion: Generic anti-mouse IgG subtype-specific antibodies can be used to improve assay sensitivity and peptides of unknown sequence can be used to quantify surrogate mAbs when the mAb protein sequence in unavailable.

RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A critical component in many of these events is the ability for RNA to interact with proteins. Over the past few decades, our understanding of such RNA-protein interactions and their importance has driven the search and development of new techniques for the identification of RNA-binding proteins. In determining which proteins bind to the RNA of interest, it is often useful to use the approach where the RNA molecule is the "bait" and allow it to capture proteins from a lysate or other relevant solution. Here, we review a collection of methods for modifying RNA to capture RNA-binding proteins. These include small-molecule modification, the addition of aptamers, DNA-anchoring, and nucleotide substitution. With each, we provide examples of their application, as well as highlight their advantages and potential challenges.