Exploring Uniform, Dual, and Dynamic Topologies of Membrane Proteins by Substituted Cysteine Accessibility Method (SCAM™).

A described simple and advanced protocol for Substituted Cysteine Accessibility Method as applied to transmembrane (TM) orientation (SCAM™) permits a topology analysis of proteins in their native state and can be universally adapted to any membrane system to either systematically map an uniform or identify and quantify the degree of mixed topology or establish transmembrane assembly dynamics from relatively static experimental data such as endpoint topologies of membrane proteins. In this approach, noncritical individual amino acids that are thought to reside in the putative extracellular or intracellular loops of a membrane protein are replaced one at the time by cysteine residue, and the orientation with respect to the membrane is evaluated by using a pair of membrane-impermeable non-detectable and detectable thiol-reactive labeling reagents. For the most water-exposed cysteine residues in proteins, the thiol pKa lies in the range of 8-9, and formation of cysteinyl thiolate ions is optimum in aqueous rather in a nonpolar environment. These features and the ease of specific chemical modification with thiol reagents are central to SCAM™. Membrane side-specific sulfhydryl labeling allows to discriminate "exposed, protected or dynamic" cysteines strategically "implanted" at desired positions throughout cysteine less target protein template. The strategy described is widely used to map the topology of membrane protein and establish its transmembrane dynamics in intact cells of both diderm (two-membraned) Gram-negative and monoderm (one-membraned) Gram-positive bacteria, cell-derived oriented membrane vesicles, and proteoliposomes.

The Mycotoxin Patulin Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier (SLC25A20) by Interaction with Cys136 Implications for Human Health.

The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated. Transport function was measured as [3H]-carnitineex/carnitinein antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Patulin (PAT) inhibited both the mitochondrial native and recombinant transporters. The inhibition was not reversed by physiological and sulfhydryl-reducing reagents, such as glutathione (GSH) or dithioerythritol (DTE). The IC50 derived from the dose-response analysis indicated that PAT inhibition was in the range of 50 µM both on the native and on rat and human recombinant protein. The kinetics process revealed a competitive type of inhibition. A substrate protection experiment confirmed that the interaction of PAT with the protein occurred within a protein region, including the substrate-binding area. The mechanism of inhibition was identified using the site-directed mutagenesis of CAC. No inhibition was observed on Cys mutants in which only the C136 residue was mutated. Mass spectrometry studies and in silico molecular modeling analysis corroborated the outcomes derived from the biochemical assays.

A Comparative Study on the Effects of the Lysine Reagent Pyridoxal 5-Phosphate and Some Thiol Reagents in Opening the Tl+-Induced Mitochondrial Permeability Transition Pore.

Lysine residues are essential in regulating enzymatic activity and the spatial structure maintenance of mitochondrial proteins and functional complexes. The most important parts of the mitochondrial permeability transition pore are F1F0 ATPase, the adenine nucleotide translocase (ANT), and the inorganic phosphate cotransporter. The ANT conformation play a significant role in the Tl+-induced MPTP opening in the inner membrane of calcium-loaded rat liver mitochondria. The present study tests the effects of a lysine reagent, pyridoxal 5-phosphate (PLP), and thiol reagents (phenylarsine oxide, tert-butylhydroperoxide, eosin-5-maleimide, and mersalyl) to induce the MPTP opening that was accompanied by increased swelling, membrane potential decline, and decreased respiration in 3 and 3UDNP (2,4-dinitrophenol uncoupled) states. This pore opening was more noticeable in increasing the concentration of PLP and thiol reagents. However, more significant concentrations of PLP were required to induce the above effects comparable to those of these thiol reagents. This study suggests that the Tl+-induced MPTP opening can be associated not only with the state of functionally active cysteines of the pore parts, but may be due to a change in the state of the corresponding lysines forming the pore structure.

Thiol-specific oxidant diamide downregulates whiA gene of Corynebacterium glutamicum, thereby suppressing cell division and metabolism.

The whiA (NCgl1527) gene from Corynebacterium glutamicum plays a crucial role during cell growth, and WhiA is recognized as the transcription factor for genes involved in cell division. In this study, we assessed the regulatory role of the gene in cell physiology. Transcription of the gene was specifically downregulated by the thiol-specific oxidant, diamide, and by heat stress. Cells exposed to diamide showed decreased transcription of genes involved in cell division and these effects were more profound in ΔwhiA cells. In addition, the ΔwhiA cells showed sensitivity to thiol-specific oxidants, DNA-damaging agents, and high temperature. Further, downregulation of sigH (NCgl0733), the central regulator in stress responses, along with master regulatory genes in cell metabolism, was observed in the ΔwhiA strain. Moreover, the amount of cAMP in the ΔwhiA cells in the early stationary phase was only at 30% level of that for the wild-type strain. Collectively, our data indicate that the role of whiA is to downregulate genes associated with cell division in response to heat or thiol-specific oxidative stress, and may suggest a role for the gene in downshifting cell metabolism by downregulating global regulatory genes when growth condition is not optimal for cells.

Ultra-rapid glutathionylation of chymotrypsinogen in its molten globule-like conformation: A comparison to archaeal proteins.

Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pKa (8.1 ± 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pKa value (7.5 ± 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the "incipit" of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present.

Isolation and analysis of a non-protein low molecular weight thiol-mercurial adduct from human prostate lymph node cells (LNCaP).

Thiol compounds present in human malignant prostate cells (LNCaP) were investigated after reaction with a mercurial blocking reagent. After extracting the cellular glutathione and some other low molecular weight (LMW) thiols using trichloroacetic acid the resulting the protein precipitate was extracted with buffered 8 M urea containing 2-chloromercuri-4-nitrophenol in an equimolar amount to that of the thiol present. After removing the insoluble chromatin fraction the urea soluble labeled adducts formed were chromatographed on G15 Sephadex. Three yellow coloured (A410 nm) fractions were obtained; first, the excluded protein fraction containing 16.0 ± 4.1% of the applied label followed by an intermediate fraction containing 5.9 ± 1.2%. Finally a LMW fraction emerged which contained 77.2 ± 3.7% of the total label applied and this was further analyzed by column chromatography, first on an anion exchange column and then on a PhenylSepharose 6 column to give what appeared to be a single component. LC-MS analysis of this component gave a pattern of mercuri-clusters, formed on MS ionization showing possible parent ions at 704 or 588 m/z, the former indicating that a thiol fragment of molecular weight approximately 467 could be present. No fragments with a single sulfur adduct (a 369 m/z fragment) were observed The adduct was analyzed for cysteine and other amino acids, nucleic acid bases, ribose and deoxyribose sugars, selenium and phosphorus; all were negative leading to the conclusion that a new class of unknown LMW thiol is present concealed in the protein matrices of these cells.

NEM assisted real-time fluorescence detection of Cys in cytoplasm and mice imaging by a Coumarin probe containing carboxyl group.

Alterations of the homeostasis balance of cysteine (Cys) are associated with a variety of diseases and cellular functions, and therefore, Cys dynamic real-time living cell intracellular imaging and quantification are important for understanding the pathophysiological processes. Thus, Cys probe that can permeate high efficiently is the first one to be affected. In fact, it is difficult for organic molecular probes to infiltrate cells because of the unique structure of the cell membrane. In this work, we found that probe containing-carboxyl just stagnated in cytomembrane due to carboxyl of probe and amino group of membrane protein forming peptide chains, nevertheless, the addition of NEM, improved membrane permeability by NEM reacting with sulfhydryl of membrane protein, which made probe permeate high efficiently and sequentially real-time detect the Cys in cytoplasm. It is the first time noted that NEM can regulate Cys probe containing-carboxyl for high efficient detection in cytoplasm. Additionally, probe was successfully applied to image Cys in mouse.

Screening of the proteins related to the cultivar-dependent cadmium accumulation of Brassica parachinensis L.

Cultivar-dependent cadmium (Cd) accumulation was principal in developing Cd-pollution safe cultivars (PSCs). Proteins related to different Cd accumulations of the low-Cd-accumulating (SJ19) and high-Cd-accumulating (CX4) cultivars were investigated by iTRAQ analysis. Higher Cd bioaccumulation factors and translocation factor in CX4 than in SJ19 were consistent with the cultivar-dependent Cd accumulations. The Cd uptake was promoted in CX4 due to its higher expression of Cd-binding proteins and the lower expression of Cd-efflux proteins in roots. What's more, significantly elevated thiol groups (PC2 and PC3) in CX4 under Cd stress might contribute to the high Cd accumulation in roots and the root-to-shoot translocation of Cd-PC complex. Up-regulated proteins involved in cellulose biosynthesis and pectin de-esterification in SJ19 enhanced the Cd sequestration of root cell walls, which was considered as the predominant strategy for reducing Cd accumulation in shoots. The present study provided novel insights in the cultivar-dependent Cd accumulation in shoots of B. parachinensis.

Design and Synthetic Strategies for Helical Peptides.

Abnormal protein-protein interactions (PPIs) are the basis of multiple diseases, and the large and shallow PPI interfaces make the target "undruggable" for traditional small molecules. Peptides, emerging as a new therapeutic modality, can efficiently mimic PPIs with their large scaffolds. Natural peptides are flexible and usually have poor serum stability and cell permeability, features that limit their further biological applications. To satisfy the clinical application of peptide inhibitors, many strategies have been developed to constrain peptides in their bioactive conformation. In this report, we describe several classic methods used to constrain peptides into a fixed secondary structure which could significantly improve their biophysical properties.

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent - dubbed 'PODS' - as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

Simple and Novel Assay of the Host-Guest Complexation of Homocysteine with Cucurbit[7]uril.

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH2CH2SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.

A Cysteine-Reactive Small Photo-Crosslinker Possessing Caged-Fluorescence Properties: Binding-Site Determination of a Combinatorially-Selected Peptide by Fluorescence Imaging/Tandem Mass Spectrometry.

To determine the binding-site of a combinatorially-selected peptide possessing a fluoroprobe, a novel cysteine reactive small photo-crosslinker that can be excited by a conventional long-wavelength ultraviolet handlamp (365 nm) was synthesized via Suzuki coupling with three steps. The crosslinker is rationally designed, not only as a bioisostere of the fluoroprobe, but as a caged-fluorophore, and the photo-crosslinked target protein became fluorescent with a large Stokes-shift. By introducing the crosslinker to a designated sulfhydryl (SH) group of a combinatorially-selected peptide, the protein-binding site of the targeted peptide was deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/fluorescence imaging followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) analysis.

Automated radiosynthesis of [18F]FBEM, a sulfhydryl site specific labeling agent for peptides and proteins.

In the process of developing [18F]FBEM coupled target peptide, we have instituted a robust automated synthesis of [18F]FBEM, a sulfhydryl (-SH) site specific agent for radiolabeling of peptides and proteins. The radiosynthesis generated 1.67-3.89 GBq (45.1-105.1 mCi, 7.5-18.8% non-decay corrected yield) of [18F]FBEM from 22.2 GBq (600 mCi) of starting [18F]fluoride with molar activity of 31.8 ±â€¯5.3 GBq/µmol (0.86 ±â€¯0.14 mCi/nmol) (n = 3) at the end of synthesis. Radiochemical purity was greater than 98%, and total synthesis time was ~90 min.

Full and partial agonists evoke distinct structural changes in opening the muscle acetylcholine receptor channel.

The muscle acetylcholine (ACh) receptor transduces a chemical into an electrical signal, but the efficiency of transduction, or efficacy, depends on the particular agonist. It is often presumed that full and partial agonists elicit the same structural changes after occupancy of their binding sites but with differing speed and efficiency. In this study, we tested the alternative hypothesis that full and partial agonists elicit distinct structural changes. To probe structural changes, we substituted cysteines for pairs of residues that are juxtaposed in the three-dimensional structure and recorded agonist-elicited single-channel currents before and after the addition of an oxidizing reagent. The results revealed multiple cysteine pairs for which agonist-elicited channel opening changes after oxidative cross-linking. Moreover, we found that the identity of the agonist determined whether cross-linking affects channel opening. For the αD97C/αY127C pair at the principal face of the subunit, cross-linking markedly suppressed channel opening by full but not partial agonists. Conversely, for the αD97C/αK125C pair, cross-linking impaired channel opening by the weak agonist choline but not other full or partial agonists. For the αT51C/αK125C pair, cross-linking enhanced channel opening by the full agonist ACh but not other full or partial agonists. At the complementary face of the subunit, cross-linking between pairs within the same ß hairpin suppressed channel opening by ACh, whereas cross-linking between pairs from adjacent ß hairpins was without effect for all agonists. In each case, the effects of cross-linking were reversed after addition of a reducing reagent, and receptors with single cysteine substitutions remained unaltered after addition of either oxidizing or reducing reagents. These findings show that, in the course of opening the receptor channel, different agonists elicit distinct structural changes.

Three unrelated and unexpected amino acids determine the susceptibility of the interface cysteine to a sulfhydryl reagent in the triosephosphate isomerases of two trypanosomes.

Proteins with great sequence similarity usually have similar structure, function and other physicochemical properties. But in many cases, one or more of the physicochemical or functional characteristics differ, sometimes very considerably, among these homologous proteins. To better understand how critical amino acids determine quantitative properties of function in proteins, the responsible residues must be located and identified. This can be difficult to achieve, particularly in cases where multiple amino acids are involved. In this work, two triosephosphate isomerases with very high similarity from two related human parasites were used to address one such problem. We demonstrate that a seventy-fold difference in the reactivity of an interface cysteine to the sulfhydryl reagent methylmethane sulfonate in these two enzymes depends on three amino acids located far away from this critical residue and which could not have been predicted using other current methods. Starting from previous observations with chimeric proteins involving these two triosephosphate isomerases, we developed a strategy involving additive mutant enzymes and selected site directed mutants to locate and identify the three amino acids. These three residues seem to induce changes in the interface cysteine in reactivity by increasing (or decreasing) its apparent pKa. Some enzymes with four to seven mutations also exhibited altered reactivity. This study completes a strategy for identifying key residues in the sequences of proteins that can have applications in future protein structure-function studies.

The therapeutic value of protein (de)nitrosylation in experimental septic shock.

Cardiovascular dysfunction and organ damage are hallmarks of sepsis and septic shock. Protein S-nitrosylation by nitric oxide has been described as an important modifier of protein function. We studied whether protein nitrosylation/denitrosylation would impact positively in hemodynamic parameters of septic rats. Polymicrobial sepsis was induced by cecal ligation and puncture. Female Wistar rats were treated with increasing doses of DTNB [5,5'-dithio-bis-(2-nitrobenzoic acid)] 30min before or 4 or 12h after sepsis induction. Twenty-four hours after surgery the following data was obtained: aorta response to phenylephrine, mean arterial pressure, vascular reactivity to phenylephrine, biochemical markers of organ damage, survival and aorta protein nitrosylation profile. Sepsis substantially decreases blood pressure and the response of aorta rings and of blood pressure to phenylephrine, as well as increased plasma levels of organ damage markers, mortality of 60% and S-nitrosylation of aorta proteins increased during sepsis. Treatment with DTNB 12h after septic shock induction reversed the loss of response of aorta rings and blood pressure to vasoconstrictors, reduced organ damage and protein nitrosylation and increased survival to 80%. Increases in protein S-nitrosylation are related to cardiovascular dysfunction and multiple organ injury during sepsis. Treatment of rats with DTNB reduced the excessive protein S-nitrosylation, including that in calcium-dependent potassium channels (BKCa), reversed the cardiovascular dysfunction, improved markers of organ dysfunction and glycemic profile and substantially reduced mortality. Since all these beneficial consequences were attained even if DTNB was administered after septic shock onset, protein (de)nitrosylation may be a suitable target for sepsis treatment.

Analysis of endogenous H2S and H2Sn in mouse brain by high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection.

Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (H2Sn), have various physiological functions in mammalian cells. Although H2Sn molecules have been considered as secondary metabolites derived from hydrogen sulfide (H2S) based on in vitro studies or predetermined reaction formula, the physiological form of BSS and their endogenous concentration remain unclear. In the present study, we aimed to improve the usual method using monobromobimane (mBB) followed by high performance liquid chromatographic (HPLC) analysis for HS－ for simultaneous determination of H2S, H2S2, H2S3 and cysteine persulfide in biological samples. We demonstrated that mBB derivatization of H2S and H2Sn standards under alkaline conditions (pH 9.5) induced significant decreases in H2S2 and H2S3 levels and a significant increase in the H2S level in an incubation time-dependent manner. Conversely, the derivatization of mBB adducts of H2S2 and H2S3 were stable under neutral conditions (pH 7.0), which is physiologically relevant. Therefore, we re-examined the method using mBB and applied an improved method for the evaluation of H2S, H2S2, and H2S3 in mouse brain under physiological pH conditions. The concentrations of H2S and H2S2 were 0.030 ± 0.004µmol/g protein and 0.026 ± 0.002µmol/g protein, respectively. Although the level of H2S3 was below the quantification limit of this method, H2S3 was detected in mouse brain. Using the method established here, we reveal for the first time the existence of endogenous H2S2 and H2S3 in mammalian brain tissues. H2S2 and H2S3 exert anti-oxidant activity and anti-carbonyl stress effects through the regulation of redox balance in neuronal cells. Thus, our observations provide novel insights into the physiological functions of BSS in the brain and into neuronal diseases involved in redox imbalance.

Discovery of Heteroaromatic Sulfones As a New Class of Biologically Compatible Thiol-Selective Reagents.

The selective reaction of chemical reagents with reduced protein thiols is critical to biological research. This reaction is utilized to prevent cross-linking of cysteine-containing peptides in common proteomics workflows and is applied widely in discovery and targeted redox investigations of the mechanisms underlying physiological and pathological processes. However, known and commonly used thiol blocking reagents like iodoacetamide, N-ethylmaleimide, and others were found to cross-react with oxidized protein sulfenic acids (-SOH) introducing significant errors in studies employing these reagents. We have investigated and are reporting here a new heteroaromatic alkylsulfone, 4-(5-methanesulfonyl-[1,2,3,4]tetrazol-1-yl)-phenol (MSTP), as a selective and highly reactive -SH blocking reagent compatible with biological applications.

Specific sequences in the N-terminal domain of human small heat-shock protein HSPB6 dictate preferential hetero-oligomerization with the orthologue HSPB1.

Small heat-shock proteins (sHSPs) are a conserved group of molecular chaperones with important roles in cellular proteostasis. Although sHSPs are characterized by their small monomeric weight, they typically assemble into large polydisperse oligomers that vary in both size and shape but are principally composed of dimeric building blocks. These assemblies can include different sHSP orthologues, creating additional complexity that may affect chaperone activity. However, the structural and functional properties of such hetero-oligomers are poorly understood. We became interested in hetero-oligomer formation between human heat-shock protein family B (small) member 1 (HSPB1) and HSPB6, which are both highly expressed in skeletal muscle. When mixed in vitro, these two sHSPs form a polydisperse oligomer array composed solely of heterodimers, suggesting preferential association that is determined at the monomer level. Previously, we have shown that the sHSP N-terminal domains (NTDs), which have a high degree of intrinsic disorder, are essential for the biased formation. Here we employed iterative deletion mapping to elucidate how the NTD of HSPB6 influences its preferential association with HSPB1 and show that this region has multiple roles in this process. First, the highly conserved motif RLFDQXFG is necessary for subunit exchange among oligomers. Second, a site ∼20 residues downstream of this motif determines the size of the resultant hetero-oligomers. Third, a region unique to HSPB6 dictates the preferential formation of heterodimers. In conclusion, the disordered NTD of HSPB6 helps regulate the size and stability of hetero-oligomeric complexes, indicating that terminal sHSP regions define the assembly properties of these proteins.

Universal and Quantitative Method To Evaluate Inhibitor Potency for Cysteinome Proteins Using a Nonspecific Activity-Based Protein Profiling Probe.

Recently, there have been a limited number of new, validated targets for small-molecule drug discovery in the pharmaceutical industry. Although there are approximately 30 000 genes in the human genome, only 2% are targeted by currently approved small-molecule drugs. One reason that many targets remain neglected by drug discovery programs is the absence of biochemical assays enabling evaluation of the potency of inhibitors in a quantitative and high-throughput manner. To overcome this issue, we developed a biochemical assay to evaluate the potency of both reversible and irreversible inhibitors using a nonspecific thiol-labeling fluorescent probe. The assay can be applied to any targets with a cysteine residue in a pocket that can accommodate small-molecule ligands. By constructing a mathematical model, we showed that the potency of compounds can be quantitatively evaluated by performing an activity-based protein profiling assay. In addition, the validity of the theory was confirmed experimentally using epidermal growth factor receptor kinase as a model target. This approach provides an assay system for targets for which biochemical assays cannot be developed. Our approach can potentially not only expand the number of exploitable targets but also accelerate the lead optimization process by providing quantitative structure-activity relationship information.