Enzyme Label Research Articles

Bioenzyme–nanoenzyme–chromogen all-in-one test strip for convenient and sensitive detection of malathion in water environment

The presence of pesticide residues in aquatic environments poses a significant threat to both aquatic ecosystems and human health. The presence of these residues can result in significant harm to aquatic ecosystems and can negatively impact the health of aquatic organisms. Consequently, this issue requires urgent attention and effective measures to mitigate its impact. However, developing sensitive and rapid detection methods remains a challenge. In this study, an all-in-one test strip, which integrated bioenzymes, nanoenzymes, and a chromogen, was developed in combination with an enzyme labeling instrument for a highly sensitive and convenient sensing of malathion residues. The oxidase activity of heme chloride (Hemin) in the strip can catalyze the oxidation of H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a blue-colored oxide. Simultaneously, the alkaline phosphatase (ALP) present in the strip can break down l-ascorbic acid-2-phosphate to produce ascorbic acid (AA). This AA then acts to reduce the oxidized form of TMB, turning it into a colorless substance and leading to the disappearance of its fluorescent signal. In the presence of a pesticide, the activity of ALP is inhibited and formation of AA is blocked, thereby preventing the reduction of oxidized TMB and producing a colored signal. According to this principle, the integrated test strip detected the target pesticide with high performance as per the optical density value determined via an enzyme marker. The detection limit of the test strip was 0.209 ng/mL with good sensitivity. The method was used for detecting malathion in actual river water samples, and the recoveries were in the range of 93.53 %–96.87 %. The newly devised technique effectively identified malathion in samples of natural water. This research has introduced a novel approach for the precise and convenient surveillance of pesticide remnants. Additionally, these discoveries could inspire the advancement of proficient multi-enzyme detection systems.

Global clean label enzyme market to reach $3.1 bn by 2034, registering a 6.1% CAGR

Global clean label enzyme market to reach $3.1 bn by 2034, registering a 6.1% CAGR

Comparison of single and double pulse laser-induced breakdown spectroscopy for the detection of biomolecules tagged with photon-upconversion nanoparticles

BackgroundLaser-induced breakdown spectroscopy (LIBS) is a well-recognized analytical technique used for elemental analysis. This method is gaining considerable attention also in biological applications thanks to its ability for spatial mapping and elemental imaging. The implementation of LIBS in the biomedical field is based on the detection of metals or other elements that either naturally occur in the samples or are present artificially. The artificial implementation of nanoparticle labels (Tag-LIBS) enables the use of LIBS as a readout technique for immunochemical assays. However, one of the biggest challenges for LIBS to meet immunoassay readout standards is its sensitivity. ResultsThis paper focuses on the improvement of LIBS sensitivity for the readout of nanoparticle-based immunoassays. First, the LIBS setup was optimized on photon-upconversion nanoparticle (UCNP) droplets deposited on the microtiter plate wells. Two collection optics systems were compared, with single pulse (SP) and collinear double pulse (DP) LIBS arrangements. By deploying the second laser pulse, the sensitivity was improved up to 30 times. The optimized SP and DP setups were then employed for the indirect detection of human serum albumin based on immunoassay with UCNP-based labels. Compared to our previous LIBS study, the detection limit was enhanced by two orders of magnitude, from 10 ng mL−1 to 0.29 ng mL−1. In addition, two other immunochemical methods were used for reference, based on the readout of upconversion luminescence of UCNPs and absorbance measurement with enzyme labels. Finally, the selectivity of the assay was tested and the practical potential of Tag-LIBS was demonstrated by the successful analysis of urine samples. Significance and noveltyIn this work, we improved the sensitivity of the Tag-LIBS method by combining new labels based on UCNPs with the improved collection optics and collinear DP configuration. In the instrumental setup optimization, the DP LIBS showed better sensitivity and signal-to-noise ratio than SP. The optimizations allowed the LIBS readout to surpass the sensitivity of enzyme immunoassay, approaching the qualities of upconversion luminescence readout, which is nowadays a state-of-the-art readout technique.

Multiplex signal amplification for ultrasensitive CRP assay via integrated electrochemical biosensor array using MOF-derived carbon material and aptamers

Accurate and precise detection of disease-associated proteins, such as C-reactive protein (CRP), remains a challenge in biosensor development. Herein, we present a novel approach－an integrated disposable aptasensor array－designed for precise, ultra-sensitive, and parallel detection of CRP in plasma samples. This integrated biosensing array platform enables multiplex parallel testing, ensuring the accuracy and reliability in sample analysis. The ultra-sensitivity of this biosensor is achieved through multiplex signal amplification. Leveraging the superior conductivity and extensive surface area of MOF-derived nanoporous carbon material (CMOF), the biosensor enhances recognition elements (aptamers) by catalyzing the horseradish peroxidase (HRP) label enzyme reaction to multiply the number of probe molecules. Optimized conditions yielded exceptional performance, exhibiting high accuracy (relative standard deviation, RSD≤10.0 %), a low detection limit (0.3 pg/mL, S/N = 3), ultra-sensitivity (0.16 μA/ng mL−1 mm−2), and a rapid response (seven parallel tests within 60 min). Importantly, this multi-unit integrated disposable aptasensor array accurately quantified CRP in human serum, demonstrating comparable results to commercial enzyme–linked immunosorbent assay (ELISA). This technology showcases promise for detecting various biomarkers using a unified approach, presenting an appealing strategy for early disease diagnosis and biological analysis.

Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine.

The development of various enzyme-linked immunosorbent assays (ELISAs) coupled with surface-enhanced Raman scattering (SERS) detection is a growing area in analytical chemistry due to their potentially high sensitivity. A SERS-based ELISA with horseradish peroxidase (HRP) as an enzymatic label, an o-phenylenediamine (oPD) substrate, and a 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such a system. However, the full capabilities of this long-known approach have yet to be revealed. The current study addresses a previously unrecognized problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of the pH, the concentration of the aggregating agent, and the particle surface chloride stabilizer were extensively evaluated. At the optimal mildly acidic pH of 3, a 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, a two orders of magnitude advantage in the limits of detection (LODs) for SERS compared to colorimetry was demonstrated for both DAP and HRP. The resulting LOD for HRP of 0.067 pmol/L (1.3 amol per assay) underscores that the developed approach is a highly sensitive technique. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

The in vivo study on antioxidant activity of wendan decoction in treating hyperlipidemia: a pharmacokinetic-pharmacodynamic (PK-PD) model.

Background: Wendan Decoction (WDD) is a six-herb Chinese medicine recipe that was first mentioned in about 652 AD. It is frequently used to treat hyperlipidemic patients' clinical complaints. According to reports, oxidative stress has a significant role in hyperlipidemia. Purpose: There has not yet been a thorough pharmacokinetic-pharmacodynamic (PK-PD) examination of the clinical efficacy of WDD in the context of hyperlipemia-related oxidative stress. Therefore, the goal of this research is to explore the antioxidant essence of WDD by developing a PK-PD model, ordering to assure its implication in treating hyperlipidemia in medical practice. Methods: The model rats of foodborne hyperlipidemia were established by feeding with high-fat feed, and the lipid-lowering effect of WDD was explored. The plasma drug concentration of rats at different doses were measured by UPL-MS/MS technology, and PK parameters were calculated using Phoenix WinNonlin 8.1 software. The level of lipid peroxide (LPO) in plasma at different time points was measured by enzyme labeling instrument. Finally, the PK-PD model was established by using Phoenix WinNonlin 8.1 software, to explore the lipid-lowering effect of WDD and the relation between the dynamic changes of chemical components and antioxidant effect. Results: The findings suggested that, WDD can reduce the levels of triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) in plasma, and high-density lipoprotein cholesterol (HDL-C) was related to the dosage. Between the peak drug levels and the WDD's maximal therapeutic response, there existed a hysteresis. WDD's effect-concentration curves displayed a counterclockwise delaying loop. Alternatively, among the ten components of WDD, hesperetin, quercetin, naringenin and tangeretin might exert more significant effects in regulating the LPO levels in hyperlipidemic rats. Conclusion: This study can be helpful for other investigators to study the lipid-lowering effect of WDD.

Entropy-driven catalysis-based lateral flow assay for sensitive detection of Alzheimer 's-associated MicroRNA

Alzheimer's disease (AD) is a degenerative disease of the brain worldwide. Currently, there is no effective cure. But accurate and early diagnosis of AD is critical to the development of patient care and future treatments. MiRNA-16 has been considered as an effective diagnostic biomarker for AD because of its regulatory effect on key proteins of AD. Herein, a colorimetric lateral flow assay (LFA) was developed for sensitive detection of miRNA-16 based on entropy-driven catalysis (EDC) amplification strategy. MiRNA-16 triggered EDC and released more linker DNAs (LDNA) of sandwich structure. Thus, AuNPs were enriched at the T-line to enhance the colorimetric signal and improve the sensitivity of visual assay. It showed good specificity and sensitivity for detecting miRNA-16 with a detection limit of 1.01 pM. The practical detection of miRNA-16 in human serum obtained satisfactory result. Significantly, EDC achieved signal amplification in homogeneous solution without enzyme and DNA labeling, leading to a cheap and easy detection of miRNA-16. Therefore, it provided a portable and rapid assay for AD-related nucleic acid, which holds a potential for point-of-care testing (POCT) of AD.

Parallel convolutional contrastive learning method for enzyme function prediction.

The function labeling of enzymes has a wide range of application value in the medical field, industrial biology and other fields. Scientists define enzyme categories by enzyme commission (EC) numbers. At present, although there are some tools for enzyme function prediction, their effects have not reached the application level. To improve the precision of enzyme function prediction, we propose a parallel convolutional contrastive learning (PCCL) method to predict enzyme functions. First, we use the advanced protein language model ESM-2 to preprocess the protein sequences. Second, PCCL combines convolutional neural networks (CNNs) and contrastive learning to improve the prediction precision of multifunctional enzymes. Contrastive learning can make the model better deal with the problem of class imbalance. Finally, the deep learning framework is mainly composed of three parallel CNNs for fully extracting sample features. we compare PCCL with state-of-art enzyme function prediction methods based on three evaluation metrics. The performance of our model improves on both two test sets. Especially on the smaller test set, PCCL improves the AUC by 2.57%. The source code can be downloaded from https://github.com/biomg/PCCL.

ИММУНОФЕРМЕНТНАЯ ТЕСТ-СИСТЕМА ДЛЯ ОЦЕНКИ НАПРЯЖЕННОСТИ ПОСТВАКЦИНАЛЬНОГО ИММУНИТЕТА К ВИРУСУ КЛАССИЧЕСКОЙ ЧУМЫ СВИНЕЙ

Classical swine fever (CSF), being a particularly dangerous transboundary disease of pigs of all breeds and ages, remains one of the most important threats to the global pig industry. An urgent task is the development of new recombinant vaccines that meet the requirements of the DIVA strategy. No less important is the issue of equipping preventive means with accurate and rapid diagnostic test systems that allow assessing the intensity of post-vaccination immunity in target animals. The purpose of this study is to construct an enzyme-linked immunosorbent test system for indicating antibodies to the CSF virus based on a recombinant antigen. Specific components of the test system were obtained using genetic engineering and biochemical methods. Optimization of the ELISA protocol was carried out on a sample of immune and normal pig sera with variations in parameters (time and temperature of sensitization, working concentrations of specific components, reaction time). Determination of the analytical characteristics of the test system included tests for specificity, sensitivity limit, positive-negative threshold, and intra-laboratory reproducibility. It was found that the following parameters are optimal for performing ELISA: sensitizing antigen concentration – 50 ng per well, sensitization time – 3 hours at 37 °C or 12 hours at 4 °C; dilution of the antispecies conjugate is 1:50000, binding time to the enzymatic label is 45 min. The analytical sensitivity limit of the test system was 190 ng/ml for protein, the intra-laboratory reproducibility of results between wells of one plate was no less than 95 %, between individual plates was no less than 93 %. The developed ELISA test system, which has high sensitivity and specificity, can be recommended for assessing the effectiveness of vaccination against CSF.

Features of immunological indicators in children with allergy modified by nickel contamination of biomedia and polymorphism of CPOX rs1131857 detoxification gene

Introduction. The impact of unfavourable environmental chemical factors on the human immune system predetermines the conditions for the formation of pathological conditions associated with allergies.
 The purpose of the study. To study the features of immunological indicator parameters in children with allergies modified by contamination of biological media with nickel and polymorphism of the detoxification CPOX rs1131857 gene.
 Materials and methods. A study was conducted one hundred eighty children suffering from various forms of allergies, permanently residing in the zone of the Far North (121 of them — under conditions of aerogenic exposure to nickel aerosols, and 59 children — in a “conditionally clean” territory). The subject of the study was biological media (blood). The change in the content of specific IgE to nickel was determined by an allergosorbent test with an enzyme label. The study of cell differentiation clusters included the use of flow cytometry (CD3+, CD19+, CD3+CD4+, CD3+CD8+, CD16+CD56+). Polymorphism of the CPOX (rs1131857) gene was studied by real-time polymerase chain reaction (PCR).
 Results. The analysis of the immune and genetic status of children with various manifestations of allergic pathology revealed cellular immunodeficiency (CD3+ CD4+, a decrease in the immunoregulatory index), the development of processes of hypersensitivity to chemical technogenic haptens (IgE to nickel), as well as an increased frequency of occurrence of heterozygous AC polymorphism of the second detoxification gene. phase CPOX rs1131857, verified by significant parameters of inheritance models (OR = 2.83; 95% CI: 1.14–7.01; 
 p < 0.05).
 Limitations. The present study is not exposed to the adult population, does not consume the population, and does not consume, except in the Far North.
 Conclusion. A feature of the course of allergic pathology in the children’s population of the Arctic, modified by contamination of biomedia with nickel and polymorphism of the detoxification CPOX rs1131857 gene, will be an increased frequency and duration of recurrence of allergopathology due to the addition of viral (decrease in cellular immunity) and hapten (nickel) components.

Application of N-Terminal Labeling Methods Provide Novel Insights into Endoproteolysis of the Prion Protein in Vivo.

Alternative α- and β-cleavage events in the cellular prion protein (PrPC) central region generate fragments with distinct biochemical features that affect prion disease pathogenesis, but the assignment of precise cleavage positions has proven challenging. Exploiting mouse transgenic models expressing wild-type (WT) PrPC and an octarepeat region mutant allele (S3) with increased β-fragmentation, cleavage sites were defined using LC-MS/MS in conjunction with N-terminal enzymatic labeling and chemical in-gel acetylation. Our studies profile the net proteolytic repertoire of the adult brain, as deduced from defining hundreds of proteolytic events in other proteins, and position individual cleavage events in PrPC α- and β-target areas imputed from earlier, lower resolution methods; these latter analyses established site heterogeneity, with six cleavage sites positioned in the β-cleavage region of WT PrPC and nine positions for S3 PrPC. Regarding α-cleavage, aside from reported N-termini at His110 and Val111, we identified a total of five shorter fragments in the brain of both mice lines. We infer that aminopeptidase activity in the brain could contribute to the ragged N-termini observed around PrPC's α- and β-cleavage sites, with this work providing a point of departure for further in vivo studies of brain proteases.

Nanoparticle-Based Bioaffinity Assays: From the Research Laboratory to the Market.

Advances in the development of new biorecognition elements, nanoparticle-based labels as well as instrumentation have inspired the design of new bioaffinity assays. This review critically discusses the potential of nanoparticles to replace current enzymatic or molecular labels in immunoassays and other bioaffinity assays. Successful implementations of nanoparticles in commercial assays and the need for rapid tests incorporating nanoparticles in different roles such as capture support, signal generation elements, and signal amplification systems are highlighted. The limited number of nanoparticles applied in current commercial assays can be explained by challenges associated with the analysis of real samples (e.g., blood, urine, or nasal swabs) that are difficult to resolve, particularly if the same performance can be achieved more easily by conventional labels. Lateral flow assays that are based on the visual detection of the red-colored line formed by colloidal gold are a notable exception, exemplified by SARS-CoV-2 rapid antigen tests that have moved from initial laboratory testing to widespread market adaption in less than two years.

Optimizing the Composition of the Substrate Enhances the Performance of Peroxidase-like Nanozymes in Colorimetric Assays: A Case Study of Prussian Blue and 3,3'-Diaminobenzidine.

One of the emerging trends in modern analytical and bioanalytical chemistry involves the substitution of enzyme labels (such as horseradish peroxidase) with nanozymes (nanoparticles possessing enzyme-like catalytic activity). Since enzymes and nanozymes typically operate through different catalytic mechanisms, it is expected that optimal reaction conditions will also differ. The optimization of substrates for nanozymes usually focuses on determining the ideal pH and temperature. However, in some cases, even this step is overlooked, and commercial substrate formulations designed for enzymes are utilized. This paper demonstrates that not only the pH but also the composition of the substrate buffer, including the buffer species and additives, significantly impact the analytical signal generated by nanozymes. The presence of enhancers such as imidazole in commercial substrates diminishes the catalytic activity of nanozymes, which is demonstrated herein through the use of 3,3'-diaminobenzidine (DAB) and Prussian Blue as a model chromogenic substrate and nanozyme. Conversely, a simple modification to the substrate buffer greatly enhances the performance of nanozymes. Specifically, in this paper, it is demonstrated that buffers such as citrate, MES, HEPES, and TRIS, containing 1.5-2 M NaCl or NH4Cl, substantially increase DAB oxidation by Prussian Blue and yield a higher signal compared to commercial DAB formulations. The central message of this paper is that the optimization of substrate composition should be an integral step in the development of nanozyme-based assays. Herein, a step-by-step optimization of the DAB substrate composition for Prussian Blue nanozymes is presented. The optimized substrate outperforms commercial formulations in terms of efficiency. The effectiveness of the optimized DAB substrate is affirmed through its application in several commonly used immunostaining techniques, including tissue staining, Western blotting assays of immunoglobulins, and dot blot assays of antibodies against SARS-CoV-2.

ENZYMES IN THE DEVELOPMENT OF PHYSICO-CHEMICAL METHODS FOR BIOMEDICAL RESEARCH

The paper addresses the possibilities of voltammetry, surface-enhanced Raman spectroscopy (SERS) and mass spectrometry in tandem with enzymatic catalysis. The basic principles of operation of electrochemical biosensors based on oxidases and dehydrogenases are described. It has been shown that biosensors using cyclic enzymatic reactions and substrate recycling have the best sensitivity. The variants of significant improvement of the analytical potential of biosensor analysis due to the use of polymers for effective modification of the electrode surface and non-destructive immobilization of enzymes are illustrated. The data demonstrating how the use of enzyme labels expands the range of bioanalytical applications of SERS are presented. The possibility of highly sensitive measurement of the activity of enzyme labels (peroxidase, alkaline phosphatase, β-galactosidase) using SERS, in fact, opens up a new universal platform for the development of methods for the determination of various antigens. By way of example of the most commonly used proteases, the main trends in the development of the methodology of proteomic studies by mass spectrometry, as well as the role of proteases in the design of mass spectrometric experiments, are considered.

An NIR Fluorescence Turn‐on and MRl Bimodal Probe for Concurrent Real‐time in vivo Sensing and Labeling of β‐Galactosidase

AbstractTo realize sensing and labeling biomarkers is quite challenging in terms of designing multimodal imaging probes. In this study, we developed a novel β‐galactosidase (β‐gal) activated bimodal imaging probe that combines near‐infrared (NIR) fluorescence and magnetic resonance imaging (MRI) to enable real‐time visualization of activity in living organisms. Upon β‐gal activation, Gal‐Cy‐Gd‐1 exhibits a remarkable 42‐fold increase in NIR fluorescence intensity at 717 nm, allowing covalent labeling of adjacent target enzymes or proteins and avoiding molecular escape to promote probe accumulation at the tumor site. This fluorescence reaction enhances the longitudinal relaxivity by approximately 1.9 times, facilitating high‐resolution MRI. The unique features of Gal‐Cy‐Gd‐1 enable real‐time and precise visualization of β‐gal activity in live tumor cells and mice. The probe's utilization aids in identifying in situ ovarian tumors, offering valuable assistance in the precise removal of tumor tissue during surgical procedures in mice. The fusion of NIR fluorescence and MRI activation through self‐immobilizing target enzymes or proteins provides a robust approach for visualizing β‐gal activity. Moreover, this approach sets the groundwork for developing other activatable bimodal probes, allowing real‐time in vivo imaging of enzyme activity and localization.

An NIR Fluorescence Turn-on and MRl Bimodal Probe for Concurrent Real-time in vivo Sensing and Labeling of β-Galactosidase.

To realize sensing and labeling biomarkers is quite challenging in terms of designing multimodal imaging probes. In this study, we developed a novel β-galactosidase (β-gal) activated bimodal imaging probe that combines near-infrared (NIR) fluorescence and magnetic resonance imaging (MRI) to enable real-time visualization of activity in living organisms. Upon β-gal activation, Gal-Cy-Gd-1 exhibits a remarkable 42-fold increase in NIR fluorescence intensity at 717 nm, allowing covalent labeling of adjacent target enzymes or proteins and avoiding molecular escape to promote probe accumulation at the tumor site. This fluorescence reaction enhances the longitudinal relaxivity by approximately 1.9 times, facilitating high-resolution MRI. The unique features of Gal-Cy-Gd-1 enable real-time and precise visualization of β-gal activity in live tumor cells and mice. The probe's utilization aids in identifying in situ ovarian tumors, offering valuable assistance in the precise removal of tumor tissue during surgical procedures in mice. The fusion of NIR fluorescence and MRI activation through self-immobilizing target enzymes or proteins provides a robust approach for visualizing β-gal activity. Moreover, this approach sets the groundwork for developing other activatable bimodal probes, allowing real-time in vivo imaging of enzyme activity and localization.

Effects of low level laser on periodontal tissue remodeling in hPDLCs under tensile stress

This study aimed to investigate the effect of Low-Level Laser Therapy (LLLT) on human Periodontal Ligament Cells (hPDLCs) under tension stress. Primary hPDLCs were obtained using the tissue culture method, and P3 cells were utilized for the subsequent experiments. The study comprised four groups: a blank control group (Group B), a laser irradiation group (Group L), a tension stress group (Group T), and a laser + tension stress group (Group LT). Mechanical loading was applied using an in-vitro cell stress loading device at a frequency of 0.5 Hz and deformation of 2% for two hours per day for two days. Laser irradiation at 808 nm GaAlAs laser was administered 1 h after force loading. Cell samples were collected after the experiment. Bone and fiber remodeling factors were analyzed using PCR and Western blot. Flow cytometry was employed to assess the cell cycle, while ROS and Ca2+ levels were measured using a multifunctional enzyme labeling instrument. The results revealed that laser intervention under tension stress inhibited the expression of osteogenic differentiation factors, promoted the expression of osteoclast differentiation factors, and significantly increased the production of collagen factors, MMPs, and TIMPs. The LT group exhibited the most active cell cycle (P < 0.05). LLLT not only enhanced Ca2+ expression in hPDLCs under tension stress, but also stimulated the production of ROS. Overall, our findings demonstrate that LLLT effectively accelerated the proliferation of hPDLCs and the remodeling of periodontal tissue, possibly through the regulation of ROS and Ca2+ levels in hPDLCs.

Ligand-Directed Chemistry on Glycoside Hydrolases - A Proof of Concept Study.

Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand-directed chemistry (LDC) for labelling glycoside hydrolases near - but not in - the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar-based reversible inhibitors for labelling of two model β-glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.

SAT012 In Vivo Mapping Of Subcellular Proteomes In Mice

Abstract Disclosure: K. Kim: None. I. Park: None. J. Kim: None. J. Kim: None. H. Rhee: None. J. Suh: None. To facilitate the understanding of metabolic changes associated with aging and disease processes we have developed new in vivo tools that enable tissue-specific profiling of subcellular proteomes. First we describe a method to profile in vivo mitochondrial proteomes utilizing transgenic mice expressing mitoAPEX, a peroxidase-based proximity labeling enzyme containing a mitochondrial matrix targeting sequence. Upon label activating conditions, mitoAPEX rapidly (&amp;lt;1 min) catalyzes production of biotin radicals which biotinylate proteins within a 20 nm radius. Mass analysis of biotinylated proteomes from proximity labeled mitoAPEX mouse tissues confirmed specific and efficient labeling of the mitochondrial proteome and revealed tissue-specific patterns of the matrix proteome. The labeled muscle proteomes from young and old mitoAPEX mice revealed significant changes in the quantity and composition of protein species. Of these, RTN4IP1, was shown to be downregulated in muscle tissue of old mice. However, in contrast to previous reports, our analysis of RTN4IP1 shows RTN4IP1 is localized to the mitochondrial matrix and not the outer membrane. In addition to the mitoAPEX mice, we generated another in vivo proximity labeling tool iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which labels secretory pathway proteins, via proximity labeling activity by ER lumen targeted TurboID, an engineered biotin ligase. We expressed iSLET in the mouse liver and demonstrate efficient labeling of the liver secreted proteome which could be tracked and identified within circulating blood plasma. We expect mitoAPEX and iSLET mice will facilitate our understanding of mitochondrial function and interorgan communication networks in aging and associated disease processes. Presentation: Saturday, June 17, 2023

Light-activated BioID - an optically activated proximity labeling system to study protein-protein interactions.

Proximity labeling with genetically encoded enzymes is widely used to study protein-protein interactions in cells. However, the accuracy of proximity labeling is limited by a lack of control over the enzymatic labeling process. Here, we present a light-activated proximity labeling technology for mapping protein-protein interactions at the cell membrane with high accuracy and precision. Our technology, called light-activated BioID (LAB), fuses the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. We demonstrate, in multiple cell lines, that upon illumination with blue light, CRY2 and CIB1 dimerize, reconstitute split-TurboID and initiate biotinylation. Turning off the light leads to the dissociation of CRY2 and CIB1 and halts biotinylation. We benchmark LAB against the widely used TurboID proximity labeling method by measuring the proteome of E-cadherin, an essential cell-cell adhesion protein. We show that LAB can map E-cadherin-binding partners with higher accuracy and significantly fewer false positives than TurboID.