Labeling Reagent Research Articles

Twelve Colors of Streptavidin-Fluorescent Proteins (SA-FPs): A Versatile Tool to Visualize Genetic Information in Single-Molecule DNA.

Streptavidin-fluorescent proteins (SA-FPs) are a versatile tool to visualize a broad range of biochemical applications on a fluorescence microscope. Although the avidin-biotin interaction is widely used, the use of SA-FPs has not been applied to single-molecule DNA visualization. Here, we constructed 12 bright SA-FPs for DNA staining or labeling reagents. To date, 810 FPs are available, many of which are brighter than organic dyes. In this study, 12 bright FPs were selected to construct SA-FP plasmids covering green to red colors. Their brightness ranges from 40 to 165 mM-1 cm-1. Moreover, SA-FP is brighter than FP itself because streptavidin forms a tetramer complex; thus, four FPs are in a single complex. In addition, FPs often form a dimer or a tetramer, resulting in multiple FPs in a single spot on a microscopic image. This feature is advantageous because multiple fluorescent β-barrels on a single biotin tag provide enough brightness to be easily visualized by epifluorescence microscopy. Using SA-FPs, we visualized DNA backbones, nickase-based optical mapping, and AT-frequency profiling. Finally, we demonstrated the combination of nickase-based optical mapping using SA-FP and AT-frequency profiling.

Combined Application of Orthogonal Sortases and Depsipeptide Substrates for Dual Protein Labeling.

Staphylococcus aureus sortase A is a transpeptidase that has been extensively exploited for site-specific modification of proteins and was originally used to attach a labeling reagent containing an LPXTG recognition sequence to a protein or peptide with an N-terminal glycine. Sortase mutants with other recognition sequences have also been reported, but in all cases, the reversibility of the transpeptidation reaction limits the efficiency of sortase-mediated labeling reactions. For the wildtype sortase, depsipeptide substrates, in which the scissile peptide bond is replaced with an ester, allow effectively irreversible sortase-mediated labeling as the alcohol byproduct is a poor competing nucleophile. In this paper, the use of depsipeptide substrates for evolved sortase variants is reported. Substrate specificities of three sortases have been investigated allowing identification of an orthogonal pair of enzymes accepting LPEToG and LPESoG depsipeptides, which have been applied to dual N-terminal labeling of a model protein mutant containing a second, latent N-terminal glycine residue. The method provides an efficient orthogonal site-specific labeling technique that further expands the biochemical protein labeling toolkit.

Overall analyses of the reactions catalyzed by acetohydroxyacid synthase/acetolactate synthase using a precolumn derivatization-HPLC method

A precolumn derivatization-HPLC method using 2,4-dinitrophenylhydrazine and 4-nitro-o-phenylenediamine as respective labeling reagents for comprehensive analyses of the reactions catalyzed by acetohydroxyacid synthase (AHAS)/acetolactate synthase (ALS) is developed and evaluated in this research. Comparison with the classic Bauerle’ UV assay which can analyze the enzymes only through measurement of acetoin production, the HPLC method shows advantages because it can analyze the enzymes not only via determination of consumption of the substrate pyruvate, but also via measurement of formation of the products including acetoin, 2,3-butanedione, and acetaldehyde in the enzymatic reactions. Thus the results deduced from the HPLC method can reflect the trait of each enzyme in a more precise manner. As far as we know, this is the first time that the reactions mediated by AHAS/ALS using pyruvate as a single substrate are globally analyzed and the features of the enzymes are properly discussed.

Living Cell-Target Responsive Accessibility Profiling Reveals Silibinin Targeting ACSL4 for Combating Ferroptosis.

We report a living cell-target responsive accessibility profiling (LC-TRAP) approach to identify the targetome of silibinin (SIL), a well-established hepatoprotective natural product (NP), in HepG2 cells. Proteins showing accessibility changes, probed by covalent lysine labeling reagents and leveraged by multiplexed quantitative proteomics, following the administration of SIL to the living cells were assigned as potential targets. Among the assigned targetome, ACSL4, an enzyme essential for ferroptosis induction, might be involved in the hepatoprotective effects of SIL and hence was intensively validated. We first demonstrated that SIL protected HepG2 cells from ferroptosis dependent on ACSL4. Then, we used biophysical assays and a SIL-derivatized chemical probe to corroborate that SIL can bind to ACSL4. The ensuing enzymatic assays showed that SIL inhibited ACSL4 enzymatic activity, thereby mitigating the ACSL4-mediated ferroptosis. As such, we revealed that ACSL4 inhibition, using SIL as a model compound, represents a promising hepatoprotective strategy. Further, since TRAP probes the accessibility changes of reactive proteinaceous lysines, it can pinpoint the proximal regions where the ligand engagement may occur. Thus, the LC-TRAP analysis of SIL, the newly discovered ligand of ACSL4, and arachidonic acid (AA), the substrate, intriguingly showed that SIL and AA both affected the conformation of the K536-proximal region of ACSL4, albeit through distinct binding patterns. Collectively, we describe a straightforward LC-TRAP workflow that does not involve ligand-derived probe synthesis and is widely applicable to target discovery of NPs.

Matrix-assisted laser desorption/ionization high-resolution mass spectrometry for high-throughput analysis of androgenic steroid adulteration in traditional Chinese medicine based on d0/d5-Girard's reagent P labeling

The illegally adulteration of traditional Chinese medicine with androgenic steroids exposes consumers to multiple health risks. Therefore, it is very important to establish an accurate detection method. The current study presents a stable isotope labeling matrix-assisted laser desorption/ionization high-resolution mass spectrometry (SIL-MALDI-HRMS) method for high-throughput quantitative analysis of androgenic steroid adulteration. A pair of isotope labeling reagents (d0/d5-Girard's reagent P, d0/d5-GP) were employed to label steroids prior to MALDI-HRMS detection. The signal intensity was significantly improved, and the signal fluctuations were compensated by employing d5-GP labeled analytes as the internal standards. The limits of quantification (LOQs) of four steroids (nandrolone, testosterone, methyltestosterone, and testosterone propionate) were in the ranges of 0.18–0.48 μg/g. The calibration curves were obtained with acceptable linearities (R2 > 0.99). The relative recoveries ranged from 87.9% to 118.1%, and the intra-day and inter-day relative standard deviations (RSDs) were all less than 13.4%, which were satisfactory for quantitative analysis. The potential of the GP labeling strategy to improve the MALDI detection sensitivity was further demonstrated by the successful detection of cinnamaldehyde and 2′-methoxycinnamaldehyde in cinnamon-containing samples. Compared with the reported methods, the developed method proved to be high-throughput, highly selective, and accurate for androgenic steroids analysis in complex samples with good sensitivity.

Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region.

The Mycobacterium tuberculosis genome harbours nine toxin-antitoxin (TA) systems of the mazEF family. These consist of two proteins, a toxin and an antitoxin, encoded in an operon. While the toxin has a conserved fold, the antitoxins are structurally diverse and the toxin binding region is typically intrinsically disordered before binding. We describe high throughput methodology for accurate mapping of interfacial residues and apply it to three MazEF complexes. The method involves screening one partner protein against a panel of chemically masked single cysteine mutants of its interacting partner, displayed on the surface of yeast cells. Such libraries have much lower diversity than those generated by saturation mutagenesis, simplifying library generation and data analysis. Further, because of the steric bulk of the masking reagent, labeling of virtually all exposed epitope residues should result in loss of binding, and buried residues are inaccessible to the labeling reagent. The binding residues are deciphered by probing the loss of binding to the labeled cognate partner by flow cytometry. Using this methodology, we have identified the interfacial residues for MazEF3, MazEF6 and MazEF9 TA systems of M. tuberculosis. In the case of MazEF9, where a crystal structure was available, there was excellent agreement between our predictions and the crystal structure, superior to those with AlphaFold2. We also report detailed biophysical characterization of the MazEF3 and MazEF9 TA systems and measured the relative affinities between cognate and non-cognate toxin–antitoxin partners in order to probe possible cross-talk between these systems.

One-Step Conversion of NHS Esters to Reagents for Site-Directed Labeling of IgG Antibodies.

Antibody conjugates are extensively used for diagnostics and therapeutics, and as a tool for molecular biology. To prepare such conjugates N-hydroxysuccinimide (NHS) esters are most often used due to the straightforward experimental procedure and the commercial accessibility of the reagents. Such conjugates are however highly heterogeneous, since only the reactivity of the lysines determines the distribution of labels. This has inspired the development of methods that experimentally are as facile but produce conjugates of higher quality. Herein, we report the development of a reagent that can, in one step, be activated with an NHS ester of choice and subsequently can be directly used for site-directed labeling of antibodies. The reagent can be prepared in three synthetic steps and produces conjugates with similar ease as for NHS esters, however in a site-directed manner. We show that the reagent is quantitatively activated by a variety of NHS esters, and we use these to functionalize IgG1, IgG2, and IgG4 antibodies.

Slow-Off-Rate-Modified Aptamer Labeling for Fluorescence Microscopy and DNA-PAINT.

Super-resolution microscopy methods enable the visualization of biological processes on the level of a few nanometers. However, the application of these techniques in biological systems is limited by the availability of small affinity reagents. Slow off-rate-modified aptamers as nucleic acid analogues to antibodies have been successfully applied to improve the resolution and quantification of DNA-PAINT. In this chapter, we describe a protocol for using SOMAmers as labeling reagents for super-resolution microscopy.

Quantitative Analysis and Structural Elucidation of Fatty Acids by Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags and m-CPBA Epoxidation.

In this study, a novel analytical method was developed to investigate fatty acids (FAs) for relative quantification, carbon-carbon double-bond localization, and cis-/trans-geometry differentiation by isobaric multiplex labeling reagents for carbonyl-containing compound (SUGAR) tag conjugation and meta-chloroperoxybenzoic acid (m-CPBA) epoxidation. FAs are essential components of cells and have diverse functions in energy storage and as complex lipid constituents. It has been reported that FAs play different roles in various biological processes such as the functional development of the brain. The comprehensive characterization and quantification of FAs are crucial to further elucidate their biological roles. However, it is challenging to perform relative quantification and structural elucidation of FAs using integrated mass spectrometry (MS)-based methods. Recently, our group developed isobaric multiplex SUGAR tags for quantitative glycomics. Besides aldehyde/ketone groups on glycans, hydrazide groups also possess reactivity toward carboxylic acids on FAs. In this study, we extended SUGAR tag labeling with FAs for the quantitative analysis by liquid chromatography (LC)-MS/MS in the positive ion mode and applied this strategy for the comparative analysis of FAs hydrolyzed from oil samples. In addition, to comprehensively elucidate the structures of unsaturated FAs, epoxidation by m-CPBA was performed before SUGAR tag labeling to enable carbon-carbon double-bond localization. Moreover, the cis- and trans-geometries of carbon-carbon double bonds in multiple pairs of monounsaturated FAs could also be differentiated in higher-energy collisional dissociation (HCD)-MS/MS. This study developed a high-throughput comprehensive FA analysis platform, which could be widely applied and utilized in biological and clinical studies.

Chemoenzymatic Labeling to Visualize Intercellular Contacts Using Lipidated SortaseA.

Methods to label intercellular contact have attracted attention because of their potential in cell biological and medical applications for the analysis of intercellular communications. In this study, a simple and versatile method for chemoenzymatic labeling of intercellularly contacting cells is demonstrated using a cell-surface anchoring reagent of a poly(ethylene glycol)(PEG)-lipid conjugate. The surface of each cell in the cell pairs of interest were decorated with sortase A (SrtA) and triglycine peptide that were lipidated with PEG-lipid. In the mixture of the two-cell populations, the triglycine-modified cells were enzymatically labeled with a fluorescent labeling reagent when in contact with SrtA-modified cells on a substrate. The selective labeling of the contacting cells was confirmed by confocal microscopy. The method is a promising tool for selective visualization of intercellularly contacting cells in cell mixtures for cell-cell communication analysis.

Evaluation and minimization of over-alkylation in proteomic sample preparation

Reduction and alkylation are essential steps in shotgun proteomic sample preparation, but over-alkylation can occur on peptide N-terminus and amino acid residues other than cysteine, which adversely affects protein identification and quantification. To date, different sample preparation protocols are used in different laboratories, but a systematic comparison of the published protocols has not been done yet in the aspect of over-alkylation. Here, we comprehensively evaluated various protocols using different denaturants, different digestion buffers and different concentrations of reduction and alkylation reagents. The suggested protocols from the manufacturers of RapiGest and ProteaseMAX induce the highest degree of over-alkylation compared with others. Carbamidomethylation at lysine increases over 4 times while using triethylammonium bicarbonate buffer compared with that using others, and it should be minimized for improving the labeling efficiency of stable isotope labeling reagents that label lysine residues. The use of digestion buffers at pH 6 reduces over-alkylation, but greatly introduces some other artificial modifications. Excessive iodoacetamide was proved to be the major cause of over-alkylation during trypsin digestion. Therefore, over-alkylation can be minimized by using low concentration of dithiothreitol and iodoacetamide for reduction and alkylation or quenching the excessive iodoacetamide before digestion.

Development and application of a sensitive phosphonium-hydrazide oligosaccharide labelling reagent in capillary electrophoresis- electrospray ionization- mass spectrometry

Glycosylation is one of the most ubiquitous post-translational modifications (PTM) of proteins. Although the ionization efficiency of native glycans is fairly low, with the assistance of chemical derivation strategies, mass spectrometry (MS) has been extensively used in glycomics because of its high sensitivity, accuracy, and speed. In this study, a novel glycan labelling reagent, (4-hydrazidebutyl) triphenylphosphonium bromide (P4HZD), with a permanent positive charge was developed. The comprehensive capabilities of P4HZD for MS analysis of oligosaccharides were evaluated in detail using maltodextrin as a standard. This labelling reagent can be used in common biological MS techniques such as matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. The MS signal intensity of maltodextrin species could be enhanced up to 96-fold in MALDI-MS by labelling with P4HZD, making P4HZD favorable for MALDI-MS-based high-throughput screening of oligosaccharides. Moreover, P4HZD-labelled oligosaccharides with a degree of polymerization (DP) from 1 to 18 could be separated and analysed by capillary electrophoresis (CE) combined with positive ion mode ESI-MS. In comparison with a commercialized oligosaccharide tag, Girard's reagent P (GirP), P4HZD was more effective for enhancing the signal of oligosaccharides in the middle or higher mass range using both ESI and MALDI ion sources. Two biologics, immunoglobulin G 2 (IgG 2) and fusion protein (FP), were chosen as model complex biological samples to test the efficacy of detection and separation of oligosaccharides by MALDI-MS and CE-ESI-MS analysis with P4HZD labelling. The results indicated that P4HZD is a promising labelling reagent for the detection of oligosaccharides in complex biological samples. The tandem workflow combines the strengths of MALDI-MS and CE-ESI-MS to fulfil the analytical demands of high-throughput screening, while affording good separation.

Novel synthesis of an [18F]aryl boronic acid ester as a reagent for 18F-labeling via Suzuki coupling

Indirect 18F labeling methods using 18F-containing compounds such as N-succinimidyl-4-[18F]fluorobenzoate and 4-[18F]fluoroiodobenzene as labeling reagents have been reported because direct 18F labeling has difficulty in labeling aromatic compounds. In this study, we synthesized the 18F-labeling reagent 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) [18F]fluorobenzene ([18F]TDBFB) using a resonant-type microwave reactor in the presence of (2,2,6,6-tetramethylpiperidine 1-oxyl and a copper catalyst. Compared with a previous report on [18F]fluorophenylboronic acid, [18F]TDBFB was synthesized simply. Moreover, we applied [18F]TDBFB for the Suzuki coupling with triflate and bromide precursors. The Suzuki coupling of [18F]TDBFB and precursors using resonant-type microwave reactor yielded 4-[18F]fluorobiphenyl and the [18F]pitavastatin derivative as the coupling products. These results show the potential of [18F]TDBFB obtained using rapid synthesis as an indirect 18F-labeling reagent.

Screening and Identification of Potential Abscisic Acid Catabolites by Chemical Labeling-Assisted Ultrahigh-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry.

In this study, a screening strategy was established based on ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry assisted by chemical isotope labeling (CIL-UPLC-HRMS) for screening and identifying abscisic acid (ABA) catabolites. Based on the structures of known ABA catabolites, this strategy first proposed the structures of catabolites to be discovered. Afterward, a pair of isotope reagents N,N-2-dimethylaminoethylamine (DMED) and d4-DMED were used as labeling reagents to label the carboxyl groups in ABA and its catabolites. Then, the mass-to-charge ratio (m/z) of DMED- and d4-DMED-labeled ABA catabolites was calculated based on the labeling schema. In light of the characteristic fragmentation patterns of the DMED-labeled standards of ABA and its catabolites, screening criteria were formulated. Using our strategy, ABA, t-ABA, and 18 ABA catabolites were identified from seven plant samples. Of the identified catabolites, 16 were known, and to our knowledge, 2 were previously unidentified. Our findings contribute to ABA catabolic network improvement.

Profiling of Urine Carbonyl Metabolic Fingerprints in Bladder Cancer Based on Ambient Ionization Mass Spectrometry.

The diagnosis of bladder cancer (BC) is currently based on cystoscopy, which is invasive and expensive. Here, we describe a noninvasive profiling method for carbonyl metabolic fingerprints in BC, which is based on a desorption, separation, and ionization mass spectrometry (DSI-MS) platform with N,N-dimethylethylenediamine (DMED) as a differential labeling reagent. The DSI-MS platform avoids the interferences from intra- and/or intersamples. Additionally, the DMED derivatization increases detection sensitivity and distinguishes carboxyl, aldehyde, and ketone groups in untreated urine samples. Carbonyl metabolic fingerprints of urine from 41 BC patients and 41 controls were portrayed and 9 potential biomarkers were identified. The mechanisms of the regulations of these biomarkers have been tentatively discussed. A logistic regression (LR) machine learning algorithm was applied to discriminate BC from controls, and an accuracy of 85% was achieved. We believe that the method proposed here may pave the way toward the point-of-care diagnosis of BC in a patient-friendly manner.

Dibenzocyclooctendiones (DBCDOs): Arginine-Selective Chemical Labeling Reagents Obtained through Benzilic Acid Rearrangement.

We demonstrate that dibenzocyclooctendiones (DBCDOs) are efficient chemical reagents for the site-specific labeling of arginine-containing biomolecules. Unlike the commonly used probes, DBCDOs undergo an irreversible ring-contracted rearrangement with the guanidinium group on arginine residues under mild reaction conditions. The regioselective dual-labeled arginine residues were obtained in a one-pot reaction with our tested substrates. The efficiency of DBCDOs reactions and their ease of synthesis make DBCDOs an attractive choice for the site-selective bioconjugation of arginine.

Determination of sulfonamide residues in cultured sea cucumber by pre-column derivatization capillary electrophoresis with fluorescence detection

A simple and mild method for the separation of sulfonamide residues based on a condensation reaction with O-phthalaldehyde solution (OPA) as labeling reagent with capillary electrophoresis has been developed. A 58.5 cm × 50 μm i.d. (50 cm effective length) untreated fused-silica capillary was used. To optimize the separation conditions, the background electrolyte concentration, pH, column temperature, voltage and other factors were evaluated. The optimal separation conditions were as follows: 20 mmol L−1 borate buffer; pH 9.1; column temperature 20 °C; separation voltage 18 kV, pressure 50 mbar and injection time 8 s. Under the optimal conditions, 10 kinds of sulfonamide derivatives could be well-separated within 8 min, and the linear ranges were 0.35–100 μg kg−1. The detection limit (at a signal-to-noise ratio of 3) was in the range of 0.12–0.25 μg kg−1, and the quantification limit (at a signal-to-noise ratio of 10) was in the range of 0.35–0.70 μg kg−1. The sulfonamide residues from cultured sea cucumber samples were determined under the optimal conditions with satisfactory results.

Abstract 1741: Cancer cell-derived extracellular vesicle mimicking: A parametric study of the physicochemical characteristics of EVs and their influence on cellular uptake in metastasis

Abstract Introduction: Extracellular vesicles (EVs) play a crucial role in disseminating cancer to distant organs, communicating with the tumor microenvironment to prepare the metastatic niche, and through horizontal transfer of oncogenic traits to recipient cells. EV parameters influencing cellular uptake include surface proteins, lipid profile, and physicochemical properties such as size and zeta potential. EVs isolated from cells are heterogeneous populations, which hinder the study of single EV variables and their influence on cellular uptake. Moreover, EV isolation is a lengthy and laborious process with an extremely low yield. Liposomes are synthetic vesicles that share properties with EVs, such as the lipid bilayer and the capability to encapsulate relevant biomolecules. In this study, we used a uveal melanoma (UM) model, a highly liver metastatic tumor, to characterize naturally occurring EVs and model them using liposomes. Methods: We utilized liposomes as a model of EVs to study how the size and zeta potential influence cellular internalization. EVs were isolated from primary UM cells (MP41) using ultracentrifugation (UC) and characterized using dynamic light scattering, nanoparticle tracking analysis, as well as electrophoretic mobility for hydrodynamic size, concentration, and zeta potential, respectively. Liposomes mimicking EVs were produced using the periodic disturbance mixer (nanoprecipitation). The lipid composition was DMPC: CHOL: DHP at different ratios and 0.2% molar concentration of SP-DiIC18(3) as labeling reagent. Liposomes were then dialyzed overnight using a membrane size of 12kDa. Cellular uptake was assessed in immortalized human hepatocytes, representing the liver microenvironment. Results: Using surface response methodology (SRM), we created a model to predict size and zeta potential depending on liposome manufacturing conditions. We produced anionic and neutral liposomes with zeta potential ~-30 mV and 0mV, respectively, and with a size of ~150 nm, similar to EVs derived from MP41 cells in order to evaluate the influence of zeta potential in cellular uptake. Our results showed no significant differences at 0 and 6 hours between anionic and neutral EV-like liposomes. By contrast, the uptake of the anionic liposomes was significantly higher at 24 h compared to neutral ones. Moreover, EV-like liposomes were produced 1000x more concentrated than naturally occurring EVs isolated using UC. Conclusion: In this study, we describe a novel approach using synthetic biology to produce EV-like liposomes as a model to study EV cellular uptake and its role in metastasis. Our data demonstrate that liposomes are a feasible tool to study EV variables individually, thereby addressing the high heterogeneity of biological samples, and producing high liposome yield, thereby helping to accelerate research Citation Format: Rubén Rodrigo López Salazar, Chaymaa Zouggari, Thupten Tsering, Prisca Bustamante Alvarez, Ion Stiharu, Catherine Mounier, Vahe Nerguizian, Julia V. Burnier. Cancer cell-derived extracellular vesicle mimicking: A parametric study of the physicochemical characteristics of EVs and their influence on cellular uptake in metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1741.

Development of quinone linked immunosorbent assay (QuLISA) based on using Folin’s reagent as a non-enzymatic tag: Application to analysis of food allergens

Enzyme-labeled immunoassays are widely used for trace analysis of clinically significant compounds owing to their high selectivity. However, utilizing an enzyme for labeling the antibody leads to many problems, including low reproducibility and inhibition of antigen-antibody immuno-reaction due to the enzymes’ instability and bulkiness, respectively. Hence, our research group has been using quinones as stable non-enzymatic tags for antibodies utilizing the redox cycle of quinones. Herein, we aimed to use the broadly available and cost-effective quinone, 1,2-naphthoquinone-4-sulfonate (NQS, Folin’s reagent), for signal tagging of immunoassays. NQS could bond with biotin in a relatively short time and without using a catalyst forming biotin-1,2-naphthoquinone (BT-NQ). The synthesized BT-NQ produces strong chemiluminescence or color upon mixing with reductant and luminol or tetrazolium salts, with sensitivity down to 7.7 nM and 49.0 nM, respectively. Next, BT-NQ was used for developing the first quinone-linked immunosorbent assay (QuLISA) through labeling biotinylated-detection antibodies using avidin and BT-NQ, and it was targeted to detect food allergen, β-casein using sandwich-immunoassay. Our method showed good linearity for determination of β-casein with good sensitivity down to 20.2 ng/mL. The results of our method were compared with ELISA kit results, and QuLISA showed good matching and higher sensitivity. Moreover, we applied Folin’s reagent for direct labeling of avidin, and the resulted compound possessed strong chemiluminescence originating from its quinone content. At last, we can conclude that the use of Folin’s reagent offered a simple, stable, sensitive, and cost-effective approach for labeling immunoassays and direct chemiluminescence labeling of proteins.

Current Advances in CETSA.

Knowing that the drug candidate binds to its intended target is a vital part of drug discovery. Thus, several labeled and label-free methods have been developed to study target engagement. In recent years, the cellular thermal shift assay (CETSA) with its variations has been widely adapted to drug discovery workflows. Western blot–based CETSA is used primarily to validate the target binding of a molecule to its target protein whereas CETSA based on bead chemistry detection methods (CETSA HT) has been used to screen molecular libraries to find novel molecules binding to a pre-determined target. Mass spectrometry–based CETSA also known as thermal proteome profiling (TPP) has emerged as a powerful tool for target deconvolution and finding novel binding partners for old and novel molecules. With this technology, it is possible to probe thermal shifts among over 7,000 proteins from one sample and to identify the wanted target binding but also binding to unwanted off-targets known to cause adverse effects. In addition, this proteome-wide method can provide information on the biological process initiated by the ligand binding. The continued development of mass spectrometry labeling reagents, such as isobaric tandem mass tag technology (TMT) continues to increase the throughput of CETSA MS, allowing its use for structure–activity relationship (SAR) studies with a limited number of molecules. In this review, we discussed the differences between different label-free methods to study target engagement, but our focus was on CETSA and recent advances in the CETSA method.