3D-Printed SERS Chips for Highly Specific Detection of Denatured Type I and IV Collagens in Blood for Early Hepatic Fibrosis Diagnosis.

Hepatic fibrosis, the insidious progression of chronic liver scarring leading to life-threatening cirrhosis and hepatocellular carcinoma, necessitates the urgent development of noninvasive and precise diagnostic methodologies. Denatured collagen emerges as a critical biomarker in the pathogenesis of hepatic fibrosis. Herein, we have for the first time developed 3D-printed collagen capture chips for highly specific surface-enhanced Raman scattering (SERS) detection of denatured type I and type IV collagen in blood, facilitating the early diagnosis of hepatic fibrosis. Employing a novel blend of denatured collagen-targeting peptide-modified silver nanoparticle probes (Ag@DCTP) and polyethylene glycol diacrylate (PEGDA), we engineered a robust ink for the 3D fabrication of these collagen capture chips. The chips are further equipped with specialized SERS peptide probes, Ag@ICTP@R1 (S-I) and Ag@IVCTP@R2 (S-IV), tailored for the targeted detection of type I and IV collagen, respectively. The SERS chip platform demonstrated exceptional specificity and sensitivity in capturing and detecting denatured type I and IV collagen, achieving detection limits of 3.5 ng/mL for type I and 3.2 ng/mL for type IV collagen within a 10-400 ng/mL range. When tested on serum samples from hepatic fibrosis mouse models across a spectrum of fibrosis stages (S0-S4), the chips consistently measured denatured type I collagen and detected a progressive increase in type IV collagen concentration, which correlated with the severity of fibrosis. This novel strategy establishes a benchmark for the multiplexed detection of collagen biomarkers, enhancing our capacity to assess the stages of hepatic fibrosis.

Universal organophosphate pesticides detection by peptide based fluorescent probes.

In practical applications, the rapid and efficient detection of universal organophosphorus pesticides (OPs) can assist inspectors in quickly identifying the presence of OPs in samples. However, this presents a challenge for most well-established methods, typically designed to detect only a specific type of organophosphorus molecule at a time. In this proof-of-concept study, we draw inspiration from the structural similarities among OPs to develop innovative peptide-based fluorescence probes for the first time, which could efficiently detect a broad range of OPs within a mere 3 min. Analysis of fluorescence curve fitting reveals a clear linear correlation between the fluorescent intensity of the peptide probes and the concentration of OPs. Additionally, the selectivity analysis indicates that these peptide fluorescent probes exhibit an excellent response to various OPs while maintaining sufficient selectivity for detecting other pesticide types. Accurate sample analysis has also highlighted the potential of these peptide probes as practical tools for the rapid detection of OPs in actual vegetable samples. In summary, this proof-of-concept study presents an innovative approach to designing and developing ultrafast, universally peptide-based OP probes. These custom-designed peptide probes may facilitate rapid sample screening and offer initial quantification for OPs, potentially saving valuable time and effort in practical OP detection.

Molecular Imaging of Diffuse Cardiac Fibrosis with a Radiotracer That Targets Proteolyzed Collagen IV.

Purpose To develop an approach for in vivo detection of interstitial cardiac fibrosis using PET with a peptide tracer targeting proteolyzed collagen IV (T-peptide). Materials and Methods T-peptide was conjugated to the copper chelator MeCOSar (chemical name, 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid) and radiolabeled with copper 64 (64Cu). PET/CT scans were acquired following intravenous delivery of 64Cu-T-peptide-MeCOSar (0.25 mg/kg; 18 MBq ± 2.7 [SD]) to male transgenic mice overexpressing ß2-adrenergic receptors with intermediate (7 months of age; n = 4 per group) to severe (10 months of age; n = 11 per group) cardiac fibrosis and their wild-type controls. PET scans were also performed following coadministration of the radiolabeled probe with nonlabeled T-peptide in excess to confirm binding specificity. PET data were analyzed by t tests for static scans and analysis of variance tests (one- or two-way) for dynamic scans. Results PET/CT scans revealed significantly elevated (2.24-4.26-fold; P < .05) 64Cu-T-peptide-MeCOSar binding in the fibrotic hearts of aged transgenic ß2-adrenergic receptor mice across the entire 45-minute acquisition period compared with healthy controls. The cardiac tracer accumulation and presence of diffuse cardiac fibrosis in older animals were confirmed by gamma counting (P < .05) and histologic evaluation, respectively. Coadministration of a nonradiolabeled probe in excess abolished the elevated radiotracer binding in the aged transgenic hearts. Importantly, PET tracer accumulation was also detected in younger (7 months of age) transgenic mice with intermediate cardiac fibrosis, although this was only apparent from 20 minutes following injection (1.6-2.2-fold binding increase; P < .05). Conclusion The T-peptide PET tracer targeting proteolyzed collagen IV provided a sensitive and specific approach of detecting diffuse cardiac fibrosis at varying degrees of severity in a transgenic mouse model. Keywords: Diffuse Cardiac Fibrosis, Molecular Peptide Probe, Molecular Imaging, PET/CT © RSNA, 2024.

Design of synthetic peptide-based fluorescence probes for turn-on detection of hyaluronan.

Herein, we designed and examined a series of fluorescent peptide-based probes for turn-on detection of hyaluronan (HA), a member of the glycosaminoglycan family. We utilized two kinds of synthetic HA-binding peptides as the binding unit for HA, and each peptide was coupled with three kinds of environment-sensitive fluorophores as the signaling unit. From the examination of the peptides, fluorophores, and the position and number of fluorophore modification, we found that X7 peptide (RYPISRPRKR) labelled with an aggregation-induced emission (AIE) fluorogen, tetraphenylethene (TPE), at the N-terminal (named TPE-X7) did function as a light-up probe for HA. The response of TPE-X7 was highly selective to higher molecular weight HA in comparison with lower ones, having the possible potential for the analysis of HA size. TPE-X7 was also applicable to the quantification of HA in synovial fluids.

Development of an environmentally sensitive fluorescent peptide probe for MrgX2 and application in ligand screening of peptide antibiotics.

Mast cells (MCs) are primary effector cells involved in immediate allergic reactions. Mas-related G protein-coupled receptor-X2 (MrgX2), which is highly expressed on MCs, is involved in receptor-mediated drug-induced pseudo-anaphylaxis. Many small-molecule drugs and peptides activate MrgX2, resulting in MC activation and allergic reactions. Although small-molecule drugs can be identified using existing MrgX2 ligand-screening systems, there is still a lack of effective means to screen peptide ligands. In this study, to screen for peptide drugs, the MrgX2 high-affinity endogenous peptide ligand substance P (SP) was used as a recognition group to design a fluorescent peptide probe. Spectroscopic properties and fluorescence imaging of the probe were assessed. The probe was then used to screen for MrgX2 agonists among peptide antibiotics. In addition, the effects of peptide antibiotics on MrgX2 activation were investigated in vivo and in vitro. The environment-sensitive property of the probe was revealed by the dramatic increase in fluorescence intensity after binding to the hydrophobic ligand-binding domain of MrgX2. Based on these characteristics, it can be used for in situ selective visualization of MrgX2 in live cells. The probe was used to screen ten types of peptide antibiotics, and we found that caspofungin and bacitracin could compete with the probe and are hence potential ligands of MrgX2. Pharmacological experiments confirmed this hypothesis; caspofungin and bacitracin activated MCs via MrgX2 in vitro and induced local anaphylaxis in mice. Our research can be expected to provide new ideas for screening MrgX2 peptide ligands and reveal the mechanisms of adverse reactions caused by peptide drugs, thereby laying the foundation for improving their clinical safety.

Peptide-anchored biomimetic interface for electrochemical detection of cardiomyocyte-derived extracellular vesicles.

Cardiomyocyte-derived extracellular vesicles (EVs) are a promising class of biomarkers that can advance the diagnosis of many kinds of cardiovascular diseases. Herein, we develop a new electrochemical method for the feasible detection of cardiomyocyte-derived EVs in biological fluids. The core design of the method is the fabrication of a peptide-anchored biomimetic interface consisting of a lipid bilayer and peptide probes. On the one hand, the lipid bilayer provides excellent antifouling ability to the electrode interface and facilitates the anchoring of peptide probes. On the other hand, the peptide probes equip the electrode interface with excellent binding capability and affinity to CD172a, a specific marker of cardiomyocyte-derived EVs, thus enabling the efficient and selective detection of target EVs. Taking EVs derived from the heart myoblast cells H9C2 as the model target, the method displays a wide linear detection range from 1 × 103 to 1 × 108 particles/mL with a desirable detection limit of 132 particles/mL. Furthermore, the method shows good performance in biological fluids such as serum, and thus may have great potential for practical use in the diagnosis of cardiovascular diseases.

Screening and identification of CD44v9 specific peptide ligand for gastric cancer detection / 西安交通大学学报(医学版)

【Objective】 To screen and verify a peptide ligand specific for CD44v9. 【Methods】 A 12-mer phage peptide library was screened on CD44v9 coated on solid phase. Candidate sequences emerged after sequencing. Candidate phages were selected using enzyme-linked immunosorbent assay. The best sequence was chosen for further study. Binding of C9-3 to CD44v overexpressed HEK-293 cells was determined using immunofluorescence. Binding affinity and specificity were verified on gastric cancer tissues with immunohistochemistry. 【Results】 Phages significantly were enriched during panning process. After sequencing, nine individual sequences occurred in 30 selected clones. Among the 9 candidate sequences, C9-3 exhibited the highest frequency. Results of ELISA showed that C9-3 had the highest OD value and selectivity. Thus, C9-3 was chosen for peptide probe synthesis. C9-3 probe stained CD44v overexpressed HEK-293 cells, but not empty vector transfected HEK-293 cells. Immunohistochemistry scores of C9-3 were significantly different between gastric cancer and paracancer tissues (t=3.953, P<0.01). A linear positive correlation was observed between C9-3 binding and CD44v9 expression (r=0.823, P<0.01). 【Conclusion】 In this study, peptide ligand of CD44v9 was successfully screened. The peptide can bind to cells and cancer tissues via CD44v9. It has potential for gastric targeting probes.

Reduced graphene oxide quenched peptide probe for caspase-8 activity detection and cellular imaging.

Cysteinyl aspartate-specific protease 8 (caspase-8) plays a key role in various biological processes by regulating apoptosis. Therefore, this makes accurate detection and intracellular imaging of caspase-8 of great importance for drug screening, disease diagnosis, and prognostication. Here, by designing a reduced graphene oxide (rGO) quenched peptide probe, we constructed a new biosensing system for monitoring in vitro and intracellular caspase-8 activity. In this system, a fluorophore-labeled peptide and rGO were used as the substrate of caspase-8 and the fluorophore quencher, respectively. The hydrolysis of caspase-8 on the polypeptide probe substrate can generate two fragments with different lengths. The release of the short fragment labeled with the fluorophore causes recovery of the fluorescence signal (Ex/Em = 520/576 nm). Under the optimized conditions, the proposed fluorescence method exhibited a linear response range of 0.2 to 5 U·mL-1 for caspase-8 with a limit of detection (LOD) of 0.2 U·mL-1 in vitro. Furthermore, this method has been successfully applied to monitoring the upregulation of intracellular caspase-8 activity caused by tert-butyl hydroperoxide (TBHP) and fluorouracil. Flow cytometry assay indicated the positive relation between the upregulation of intracellular caspase-8 activity and cell apoptosis rate. In summary, the above results demonstrated the practical application of this method for apoptosis-related cell imaging.

4-Dimethylaminoantipyrine as a Broad Electrochemical Indicator for Immunosensors Platform.

Here, we describe 4-dimethylaminoantipyrine (4-DMAA)-mediated interfacing as a broad biochemical indicator to stabilize and promote the higher response of electrodes for immunological detection. We hypothesized that the improved biological interactions of 4-DMAA with electrodes and biological samples may be due to the interaction properties of the benzene and pyrazole chemical groups with graphite and proteins, respectively. In order to demonstrate that 4-DMAA could be used as a general indicator in electrochemical immunoassays, we used peptides as probes for the diagnosis of four neglected tropical infectious diseases Tegumentary leishmaniasis, Visceral leishmaniasis, Strongyloidiasis, and Leprosy on commercial graphite screen-printed electrodes. 4-DMAA oxidation was used to indicate specific biological recognition between the epitope-based peptide and serum immunoglobulin G (IgG) from infected patients. We demonstrated that 4-DMAA should be incorporated into the electrodes prior to serum application, which avoids interference with its sensitivity and specificity. In addition, 4-DMAA oxidizes at a low anodic potential, and the oxidation peak is useful for detecting proteins in biological fluids. In summary, we have successfully demonstrated the broad application of 4-DMAA as a general indicator for the specific diagnosis of four infectious diseases in electrochemical immunosensors. Such a strategy is quite advantageous for indirect detection of proteins that lack electrochemical activities or are spatially inaccessible on the electrode surface. This new indicator opens a new avenue for monitoring biological recognition, especially for immunosensors.

Synthesis and biological evaluation of Al[18F]-NOTA-IPB-PDL1P as a molecular probe for PET imaging of PD-L1 positive tumors.

PD-L1 is widely expressed in a variety of tumors, including NSCLC, melanoma, renal cell carcinoma, gastric cancer, hepatocellular as well as cutaneous and various leukemias, multiple myeloma and so on. Herein, we designed a novel peptide imaging agent (Al[18F]-NOTA-IPB-PDL1P) that specifically targets PD-L1 expressed in tumors. The overall radiochemical yield of Al[18F]-NOTA-IPB-PDL1P from 18F- was 10-15% (corrected radiochemical yield) within 20 min and the radiochemical purity of Al[18F]-NOTA-IPB-PDL1P was > 95% with a molar activity of 44.4-64.8 GBq/µmol. The lipophilicity logP value of Al[18F]-NOTA-IPB-PDL1P at pH 7.4 was -1.768 ±â€¯0.007 (n = 3). In the cellular uptake experiment, both HCT116 and PC3 cells dispalyed high uptake to Al[18F]-NOTA-IPB-PDL1P. The results of biodistribution showed that the uptake of Al[18F]-NOTA-IPB-PDL1P was high in kidneys, gall bladder and lung, and low in muscle and brain. In vivo micro PET studies, both HCT116 and PC3 tumors displayed high uptake for Al[18F]-NOTA-IPB-PDL1P, the tumor/muscle (T/M) radio was 2.93 and 3.57 respectively at 120 min. All the results indicate that Al[18F]-NOTA-IPB-PDL1P may have potential to be a PET imaging agent of tumors with high PD-L1 expression.

Serum Metal Ion-Induced Cross-Linking of Photoelectrochemical Peptides and Circulating Proteins for Evaluating Cardiac Ischemia/Reperfusion.

Patients having experienced the ischemia-reperfusion process are particularly vulnerable to subsequent heart attacks because this process can induce myocardial fibrosis, hallmarked by the release of reactive oxygen species and some proteases, such as cathepsin G, into the circulating blood. If these risk indicators can be monitored from the peripheral serum, early diagnosis and intervention may become a reality. For this purpose, we have designed an assay of free copper ions and cathepsin G in serum using only synthetic small molecules as the biosensing elements. No antibodies are needed to recognize the target protein, and no enzymes are needed to generate and amplify the biosensing signal. In this design, a short peptide can target-specifically recognize protease, while the copper ion in the serum can stimulate the photoelectrochemical activity of the probe, resulting in cross-linking of the serum proteins in a target protein-specific manner. Using this method, serum cathepsin G and free copper ion are found to be significantly elevated in the blood samples collected from patients with acute myocardial infarction and successful percutaneous coronary intervention in comparison with healthy controls, indicating a higher risk of subsequent myocardial injury and cardiovascular events. These results may point to the possible application of the proposed assay to evaluate the severity and prognosis of cardiac ischemia/reperfusion in the near future.

Development and preliminary evaluation of an integrin α2ß1-targeted PET probe as a supplement and alternative of PSMA imaging for prostate cancer.

An integrin α2ß1-targeted PET probe (68Ga-IABtP) was developed to serve as a supplement and alternative of PSMA imaging for prostate cancer. 68Ga-IABtP was synthesized by labeling the precursor peptide with 68Ga with 93% labeling yield and 4.14 MBq/µg specific radioactivity. 68Ga-IABtP showed no specific uptake in LNCaP prostate cancer cell with low integrin α2ß1 expression but significantly increased uptake in PC-3 prostate cancer cell with high integrin α2ß1 expression, which could be specifically blocked by the integrin α2ß1 monoclonal antibody. The efflux experiments demonstrated that 68Ga-IABtP could rapidly penetrate into PC-3 cell after cell binding, thereby prolonging the residence time in the tumor and allow enough time for probe clearance from the circulation and non-specific organs. The biodistribution study indicated that 68Ga-IABtP showed no specific accumulation in non-target organs and was quickly cleared from the kidney. The in vivo PET-CT imaging demonstrated that 68Ga-IABtP showed no specific uptake in LNCaP tumor but could specifically accumulate in the PC-3 tumor, and was rapidly cleared from spleen, intestine, kidney and liver, resulting in excellent contrast effect with low background signal and high target to non-target ratios.

Peptide probes containing a non-hydrolyzable phosphotyrosine-mimetic residue for enrichment of protein tyrosine phosphatases.

We developed peptide probes containing a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F2 Pmp) for the enrichment of protein tyrosine phosphatases (PTPs). We found that different F2 Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F2 Pmp failed to enrich PTPs due to dephosphorylation during the pulldown step. This enrichment approach using peptides containing F2 Pmp could be a generic tool for tyrosine phosphatome analysis without the use of antibodies.

Highly selective fluorescence probe with peptide backbone for imaging mercury ions in living cells based on aggregation-induced emission effect.

Mercury is an anthropogenic toxic heavy metal found in the environment. It is highly desirable to develop a fluorescence probe that can selectively and sensitively detect mercury ions using a turn-on response. This paper reports the successful development of a peptide fluorescence probe, TP-2 (TPE-Trp-Pro-Gln-His-Glu-NH2), which uses aggregation-induced emission effects and high selectivity to detect Hg2+. After fluorescence was activated, Hg2+ was efficiently detected using the change in fluorescence intensity. The detection limit for Hg2+ in the buffer solution was 41 nM (R2 = 0.9952). Owing to its high sensitivity, high cell permeability, and low biotoxicity, the probe could perform live cell imaging under biological conditions. This study demonstrated that TP-2 can detect Hg2+ in complex biological environments.

Electrochemical Resonance of Molecular Motion Enabling Label-, Antibody-, and Enzyme-Free Detection of SARS-CoV-2.

In this work, we developed a method to detect two viral marker proteins, the main protease and the spike protein (S protein), of SARS-CoV-2, as well as a host marker, chemokine receptor 5 (CCR5), which is associated with the risk of developing the severe acute respiratory syndrome. This assay can be completed in two steps in a label-free fashion, yielding a "signal-on" signal readout, which usually cannot be attained by electrochemical label-free detection using no labels or markers to tag the target protein. The proposed assay also utilizes no antibodies or enzyme-based reagents. The method achieves this performance by moderating the frequency of electrochemical potential scanning such that the scanning rate keeps pace with, or "resonances" with, the molecular motion of the probe molecule. This method has been successfully applied to detect the three target proteins in serum samples collected from patients infected with SARS-CoV-2, and the results indicate a strong correlation with the risk of deteriorating into severe acute conditions after virus infection. Soon, the clinical application of this method may provide a low-cost but effective method for virus surveillance in the general public.

Development of a Noninvasive KIM-1-Based Live-Imaging Technique in the Context of a Drug-Induced Kidney-Injury Mouse Model.

The development of reliable methods to diagnose acute kidney injury is essential to allow the adoption of early therapeutic interventions and evaluate their effectiveness. Based on the fact that kidney injury molecule-1 (KIM-1) expression levels in kidneys are markedly upregulated early after a damage event, here we developed a noninvasive KIM-1-based molecular imaging technique to detect kidney injury. First, we took advantage of a phage-display platform to select small peptides demonstrating a specific high binding affinity to KIM-1. The promising candidate was conjugated with fluorescent probes, and its imaging potential was validated in vitro and in vivo. This peptide, with the sequence CNRRRA, not only showed a high imaging potential in vitro, allowing a strong detection of KIM-1 expressing cells by microscopy and flow cytometry but also generated a strong kidney-specific signal in live-imaging in vivo experiments in the context of a drug-induced kidney-injury mouse model. Our data overall suggest that the CNRRRA peptide is a promising probe to use in the context of in vivo imaging for the detection of KIM-1 overexpression in damaged kidneys.

Screening and identification of a CD44v6 specific peptide using improved phage display for gastric cancer targeting.

BACKGROUND: Peptide probes can be applied for biomarker targeting to improve the diagnostic accuracy. Cluster of differentiation 44 (CD44) is up-regulated in gastric cancer (GC). Among all the variants of CD44, CD44v6 is reported the most promising biomarker for GC. The purpose of this study was generating and identification a peptide ligand specific to CD44v6. METHODS: A 12-mer phage peptide library was screened on CD44v overexpressed HEK-293 cells with an improved subtractive method. Five candidate sequences emerged. Candidate phages were selected using enzyme-linked immunosorbent assay and competitive inhibition assays. Then the sequence (designated ELT) was chosen for further study. Its binding affinity and specificity were verified on recombinant protein, GC cells, GC tissues and xenograft models based on BALB/c-nu/nu mice using dissociation constant calculation, immunofluorescence, immunohistochemistry and in vivo imaging separately. RESULTS: The dissociation constant of ELT with recombinant protein was 611.2 nM. ELT stained CD44v overexpressed HEK-293 but not the cell expressing wild-type CD44s. On GC cell lines, ELT co-stained with anti-CD44v6 antibody. ELT binding on tumor tissues significantly increased compared with that of paracancer tissues, also showed a linear positive correlation with CD44v6 expression. ELT specifically accumulated in tumor and eliminated in short time in vivo. CONCLUSIONS: ELT can target GC in vitro and in vivo via CD44v6, indicating its potential to serve as a probe for GC targeting diagnosis and therapy.

Matrix metalloproteinase 9-activatable peptide-conjugated hydrogel-based fluorogenic intraocular-lens sensor.

The eye is an extension of the central nervous system (CNS) and contains aqueous humor (AH), which is a fluid rich in biomolecules secreted from intraocular tissues; thus, this organ allows for non-invasive visualization of early changes in CNS disorders. There is a growing interest in developing implantable devices, such as intraocular-lens (IOL), for specific medical uses, including intraocular monitoring. We describe a novel IOL-sensing system for detecting AH biomarkers via biocompatible enzyme-activatable fluorogenic hydrogel sensors. Matrix-metalloproteinase-9, a biomarker of degenerative CNS and eye disorders, was selected as a target. A peptide-probe-incorporated fluorogenic IOL (FIOL) was developed using diacrylamide-group-modified poly(ethyleneglycol) (PEGDAAm) biocompatible hydrogels, adjusting the hydrogel mesh size to allow selective penetration of the target while blocking non-targets, using label-free detection with semi-permanently implantable sensors, and demonstrating the clinical feasibility of FIOL through in vivo testing. This novel FIOL-based sensing system represents a promising approach for liquid biopsy of intraocular fluids.

A photocleavable and mass spectrometric DNA-peptide probe enables fast and specific enzyme-free detection of microRNA.

MiRNAs are known to be involved in a series of diseases, including breast cancer, and they have the potential to serve as diagnostic/prognostic markers and therapeutic targets. A prerequisite for miRNAs to be applied in clinical practice is the quantitative profiling of their expression. However, the majority of current assays used in miRNA detection are highly enzyme-dependent. In this study, a novel enzyme-free assay was developed that relies on stacking hybridization and a photocleavable DNA-PL-peptide probe, which contains a reporter peptide (AVLGVDPFR), a photocleavable o-nitrobenzyl derivative linker and a detection DNA sequence that is complementary to a part of the target miRNA (e.g., miR-21, miR-125a or miR-200c). Stacking hybridization enabled the DNA-PL-peptide probe to capture DNA in a contiguous tandem arrangement to generate a long DNA single strand complementary to the target miRNA. Then, photolysis was initiated to rapidly release the reporter peptide, and the reporter peptide was ultimately monitored by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this experiment, the parameters linked with photorelease, binding, conjugation and hybridization were characterized. The results showed that the assay time was significantly shortened, and the detection specificity was improved. After validation of the assay, the target miRNA level was determined in human breast cells and tissue samples. The results demonstrated that photocleavable materials coupled with mass spectrometric detection have great potential in clinical practice.

Cyclization Reaction-Based Turn-on Probe for Covalent Labeling of Target Proteins.

Fluorescent molecules have contributed to basic biological research but there are currently only a limited number of probes available for the detection of non-enzymatic proteins. Here, we report turn-on fluorescent probes mediated by conjugate addition and cyclization (TCC probes). These probes react with multiple amino acids and exhibit a 36-fold greater emission intensity after reaction. We analyzed the reactions between TCC probes and nuclear receptors by electrospray ionization mass spectrometry, X-ray crystallography, spectrofluorometry, and fluorescence microscopy. In vitro analysis showed that probes consisting of a protein ligand and TCC could label vitamin D receptor and peroxisome proliferator-activated receptor γ. Moreover, we demonstrated that not only a ligand unit but also a peptide unit can label the target protein in a complex mixture.