Pub Date : 2025-02-20DOI: 10.1021/acs.analchem.4c05454
Xian Wang, Deyu Yi, Mengyuan Li, Zhengping Li
The DNA repair system relies on the coordinated action of multiple enzymes to maintain genomic stability, with apurinic/apyrimidinic endonuclease 1 (APE1) and flap endonuclease 1 (FEN1) playing pivotal roles in the long-patch base excision repair (LP-BER) pathway. Elevated levels of APE1 and FEN1 have been associated with tumor progression and resistance to therapy, making them key biomarkers for cancer diagnosis and treatment monitoring. Here, we present a sequentially activated AND-logic DNA sensor (D-AF) for the correlated imaging of APE1 and FEN1 in living cells. The sensor operates through a sequential process: APE1 first recognizes and cleaves an apurinic site, initiating structural changes that enable FEN1 to cleave a 5′ flap, resulting in restored fluorescence. We demonstrate the use of the D-AF-based nanosensor for in situ imaging of APE1 and FEN1 activities in cancer cells and for monitoring of enzyme dynamics during chemotherapy. This platform offers a valuable tool for investigating DNA repair mechanisms and their roles in cancer diagnosis and treatment.
{"title":"Sequential Activation of DNA Sensor Enables Correlated Imaging of Dual-Enzyme Activities in Living Cells","authors":"Xian Wang, Deyu Yi, Mengyuan Li, Zhengping Li","doi":"10.1021/acs.analchem.4c05454","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05454","url":null,"abstract":"The DNA repair system relies on the coordinated action of multiple enzymes to maintain genomic stability, with apurinic/apyrimidinic endonuclease 1 (APE1) and flap endonuclease 1 (FEN1) playing pivotal roles in the long-patch base excision repair (LP-BER) pathway. Elevated levels of APE1 and FEN1 have been associated with tumor progression and resistance to therapy, making them key biomarkers for cancer diagnosis and treatment monitoring. Here, we present a sequentially activated AND-logic DNA sensor (D-AF) for the correlated imaging of APE1 and FEN1 in living cells. The sensor operates through a sequential process: APE1 first recognizes and cleaves an apurinic site, initiating structural changes that enable FEN1 to cleave a 5′ flap, resulting in restored fluorescence. We demonstrate the use of the D-AF-based nanosensor for <i>in situ</i> imaging of APE1 and FEN1 activities in cancer cells and for monitoring of enzyme dynamics during chemotherapy. This platform offers a valuable tool for investigating DNA repair mechanisms and their roles in cancer diagnosis and treatment.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"29 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143462228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pyrophosphate (PPi) and nucleic acid amplification play a critical role in medical diagnostics, making the development of precise nanosensors essential. Lanthanide metal–organic frameworks (Ln-MOFs) are increasingly recognized for their potential in advanced luminescent biosensing applications. However, research on customized controllable responses in Ln-MOF nanosensors is still lacking, which is critical for the molecular-level modular design. In this work, we introduce a ligand engineering strategy to regulate coordination-induced antenna effect emission in Ln-MOFs, optimizing their pyrophosphate (PPi) sensing from fluorescence turn-off to turn-on modes. By tuning the coordination environment through ligand programming, we discovered a “near coordination compensation” effect, allowing for controllable transitions between aggregation-induced emission and quenching (AIE/AIQ). This reversible response was supported by density functional theory calculations. Using a Eu3+/Tb3+ dual-emission Ln-MOF designed with 2,6-pyridinedicarboxylic acid as the optimized ligand, we developed a self-correcting PPi nanosensor with a detection limit of 0.2 μM. Moreover, this system enabled ultrasensitive nucleic acid detection, achieving a limit of detection (LOD) as low as 1 fM, with applications in DNA pyrosequencing, qPCR, and DNA epigenetic modification (5-formylcytosine) analysis. These findings shed light on the structural and photophysical factors controlling Ln-MOF luminescence, offering a promising platform for highly accurate and sensitive nucleic acid detection in biomedical diagnostics.
{"title":"Customized Controllable Pyrophosphate Nanosensor Based on Lanthanide Metal–Organic Frameworks for Accurate and Sensitive Detection of Nucleic Acids","authors":"Long Yu, Yumin Feng, Qianqian Yuan, Shuang Peng, Yuxiu Xiao, Gaosong Wu, Xiang Zhou","doi":"10.1021/acs.analchem.4c06590","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06590","url":null,"abstract":"Pyrophosphate (PPi) and nucleic acid amplification play a critical role in medical diagnostics, making the development of precise nanosensors essential. Lanthanide metal–organic frameworks (Ln-MOFs) are increasingly recognized for their potential in advanced luminescent biosensing applications. However, research on customized controllable responses in Ln-MOF nanosensors is still lacking, which is critical for the molecular-level modular design. In this work, we introduce a ligand engineering strategy to regulate coordination-induced antenna effect emission in Ln-MOFs, optimizing their pyrophosphate (PPi) sensing from fluorescence turn-off to turn-on modes. By tuning the coordination environment through ligand programming, we discovered a “near coordination compensation” effect, allowing for controllable transitions between aggregation-induced emission and quenching (AIE/AIQ). This reversible response was supported by density functional theory calculations. Using a Eu<sup>3+</sup>/Tb<sup>3+</sup> dual-emission Ln-MOF designed with 2,6-pyridinedicarboxylic acid as the optimized ligand, we developed a self-correcting PPi nanosensor with a detection limit of 0.2 μM. Moreover, this system enabled ultrasensitive nucleic acid detection, achieving a limit of detection (LOD) as low as 1 fM, with applications in DNA pyrosequencing, qPCR, and DNA epigenetic modification (5-formylcytosine) analysis. These findings shed light on the structural and photophysical factors controlling Ln-MOF luminescence, offering a promising platform for highly accurate and sensitive nucleic acid detection in biomedical diagnostics.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"50 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143452153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Extracellular vesicles (EVs) are crucial mediators in various physiological and pathological processes, facilitating intercellular communication and offering potential as diagnostic disease markers. However, existing EVs separation methods have limitations that hinder their clinical application. In this study, we present a novel approach using bifunctional silica microspheres (SiO2-PTB-PS) for the specific, nondestructive isolation of EVs from complex biological media. The isolated EVs were subsequently used for direct cancer detection in clinical samples. The SiO2-PTB-PS microspheres, functionalized with a phosphatidylserine (PS) recognition peptide (PSpep), specifically bound to PS on the EVs surface. Additionally, an anti-adhesion coating on the silica microspheres minimized protein contamination, enhancing purity. This affinity-based recognition and antifouling strategy ensured high-purity EVs separation. Furthermore, we developed a detection system combining SiO2-PTB-PS microspheres with surface-enhanced Raman scattering (SERS) nanoprobes to identify protein tyrosine kinase 7 (PTK7) and epithelial cell adhesion (EpCAM) on the EVs membrane, achieving 80% precision in distinguishing cancer patients from healthy donors. The SiO2-PTB-PS microsphere system shows significant promise as a biotechnology tool, advancing the clinical application of EVs-based diagnostics.
{"title":"Extracellular Vesicles Separation and Biomedical Application Based on Affinity Recognition and Antifouling Coating Bifunctional Microsphere","authors":"Yuxing He, Jia Kang, Xuwen Yang, Nan Deng, Lingyun Hui, Yunxuan Yu, Yangyang Bian, Fufang Tao, Xinrui Duan, Jing Zhang","doi":"10.1021/acs.analchem.4c06347","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06347","url":null,"abstract":"Extracellular vesicles (EVs) are crucial mediators in various physiological and pathological processes, facilitating intercellular communication and offering potential as diagnostic disease markers. However, existing EVs separation methods have limitations that hinder their clinical application. In this study, we present a novel approach using bifunctional silica microspheres (SiO<sub>2</sub>-PTB-PS) for the specific, nondestructive isolation of EVs from complex biological media. The isolated EVs were subsequently used for direct cancer detection in clinical samples. The SiO<sub>2</sub>-PTB-PS microspheres, functionalized with a phosphatidylserine (PS) recognition peptide (PSpep), specifically bound to PS on the EVs surface. Additionally, an anti-adhesion coating on the silica microspheres minimized protein contamination, enhancing purity. This affinity-based recognition and antifouling strategy ensured high-purity EVs separation. Furthermore, we developed a detection system combining SiO<sub>2</sub>-PTB-PS microspheres with surface-enhanced Raman scattering (SERS) nanoprobes to identify protein tyrosine kinase 7 (PTK7) and epithelial cell adhesion (EpCAM) on the EVs membrane, achieving 80% precision in distinguishing cancer patients from healthy donors. The SiO<sub>2</sub>-PTB-PS microsphere system shows significant promise as a biotechnology tool, advancing the clinical application of EVs-based diagnostics.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"15 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143452154","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DNA nanostructures have recently attracted more attention with functionalities, programmability, and biocompatibility. Herein, a novel self-assembled photocatalytic DNA/SYBR Green I (SG-I) nanoflower (DSNF) was successfully synthesized by rolling circle amplification. DSNF was self-assembled through liquid crystallization of a high concentration of DNA in the RCA products, without relying on the Watson–Crick base-pairing principle. Interestingly, DSNF not only possessed a larger specific surface area and good stability but also exhibited excellent photocatalytic activity that generates singlet oxygen and superoxide anion to oxidate 3,3′,5,5′-tetramethylbenzidine. Meanwhile, the photocatalytic DSNF combined with an enzyme-linked immunosorbent assay to develop a new colorimetric sensor for highly specific, sensitive, and visual detection of carcinoembryonic antigens (CEAs). The colorimetric sensor achieved sensitive and low-cost quantitative detection of CEA in the linear range of 0.5–80.0 ng/mL, and the LOD was 0.5 ng/mL. In addition, three negative and seven positive clinical serum samples of CEA were obtained with 100% accuracy using the proposed colorimetric sensor, showing great potential in the clinical application of cancer diagnosis. We envision that this photocatalytic DSNF is expected to provide important perspectives in fluorescence imaging, photosensitizing cancer therapy, and clinical diagnosis fields.
{"title":"Self-Assembled DNA/SG-I Nanoflower: Versatile Photocatalytic Biosensors for Disease-Related Markers","authors":"Shan He, Yiyu Chen, Huiting Lian, Xuegong Cao, Bin Liu, Xiaofeng Wei","doi":"10.1021/acs.analchem.4c04772","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c04772","url":null,"abstract":"DNA nanostructures have recently attracted more attention with functionalities, programmability, and biocompatibility. Herein, a novel self-assembled photocatalytic DNA/SYBR Green I (SG-I) nanoflower (DSNF) was successfully synthesized by rolling circle amplification. DSNF was self-assembled through liquid crystallization of a high concentration of DNA in the RCA products, without relying on the Watson–Crick base-pairing principle. Interestingly, DSNF not only possessed a larger specific surface area and good stability but also exhibited excellent photocatalytic activity that generates singlet oxygen and superoxide anion to oxidate 3,3′,5,5′-tetramethylbenzidine. Meanwhile, the photocatalytic DSNF combined with an enzyme-linked immunosorbent assay to develop a new colorimetric sensor for highly specific, sensitive, and visual detection of carcinoembryonic antigens (CEAs). The colorimetric sensor achieved sensitive and low-cost quantitative detection of CEA in the linear range of 0.5–80.0 ng/mL, and the LOD was 0.5 ng/mL. In addition, three negative and seven positive clinical serum samples of CEA were obtained with 100% accuracy using the proposed colorimetric sensor, showing great potential in the clinical application of cancer diagnosis. We envision that this photocatalytic DSNF is expected to provide important perspectives in fluorescence imaging, photosensitizing cancer therapy, and clinical diagnosis fields.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"12 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143452150","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-19DOI: 10.1021/acs.analchem.4c04516
Yan Sun, Damian Houde, Roxana E. Iacob, Jason Baird, Robert V. Swift, Michael Holliday, Xuyan Shi, Simone Sidoli, Michael Brenowitz
Characterizing protein–ligand interactions is crucial to understanding cellular metabolism and guiding drug discovery and development. Herein, we explore complementing hydrogen/deuterium exchange mass spectrometry (HDX-MS) with a recently developed Fenton chemistry-based approach to protein oxidative footprinting mass spectrometry (OX-MS) to discriminate the binding of small-molecule therapeutics. Using drug-dependent perturbation as the experimental report, this combination of techniques more clearly differentiates the in-solution binding profiles of Venetoclax (ABT-199, GDC-0199-AbbVie and Genentech) and a drug candidate S55746 (Servier) to the apoptotic regulatory protein Bcl-2 than either technique alone. These results highlight the value of combining these methods to compare compounds in drug discovery and development. To better understand the structural context of the HDX-MS and OX-MS drug-dependent perturbations, we mapped these data on Bcl-2-Venetoclax and Bcl-2-S55746 cocrystal structures and compared these results with the structure of apo Bcl-2. HDX-MS shows that Venetoclax more strongly impacts the protein backbone compared to S55746. OX-MS reveals oxidation perturbations rationalized by direct side-chain protection as well as by crystallographically observed drug-induced protein restructuring. Both methods report the perturbation of some, but not all, residues mapped within 4 Å of the bound drugs in the crystal structures. Concordant characterization of backbone and side-chain accessibility will enhance our understanding of in-solution protein structure dynamics and protein–ligand interactions during drug discovery, development, and characterization, particularly when high-resolution structures are lacking.
{"title":"Hydrogen/Deuterium Exchange and Protein Oxidative Footprinting with Mass Spectrometry Collectively Discriminate the Binding of Small-Molecule Therapeutics to Bcl-2","authors":"Yan Sun, Damian Houde, Roxana E. Iacob, Jason Baird, Robert V. Swift, Michael Holliday, Xuyan Shi, Simone Sidoli, Michael Brenowitz","doi":"10.1021/acs.analchem.4c04516","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c04516","url":null,"abstract":"Characterizing protein–ligand interactions is crucial to understanding cellular metabolism and guiding drug discovery and development. Herein, we explore complementing hydrogen/deuterium exchange mass spectrometry (HDX-MS) with a recently developed Fenton chemistry-based approach to protein oxidative footprinting mass spectrometry (OX-MS) to discriminate the binding of small-molecule therapeutics. Using drug-dependent perturbation as the experimental report, this combination of techniques more clearly differentiates the in-solution binding profiles of Venetoclax (ABT-199, GDC-0199-AbbVie and Genentech) and a drug candidate S55746 (Servier) to the apoptotic regulatory protein Bcl-2 than either technique alone. These results highlight the value of combining these methods to compare compounds in drug discovery and development. To better understand the structural context of the HDX-MS and OX-MS drug-dependent perturbations, we mapped these data on Bcl-2-Venetoclax and Bcl-2-S55746 cocrystal structures and compared these results with the structure of apo Bcl-2. HDX-MS shows that Venetoclax more strongly impacts the protein backbone compared to S55746. OX-MS reveals oxidation perturbations rationalized by direct side-chain protection as well as by crystallographically observed drug-induced protein restructuring. Both methods report the perturbation of some, but not all, residues mapped within 4 Å of the bound drugs in the crystal structures. Concordant characterization of backbone and side-chain accessibility will enhance our understanding of in-solution protein structure dynamics and protein–ligand interactions during drug discovery, development, and characterization, particularly when high-resolution structures are lacking.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"14 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143443868","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-19DOI: 10.1021/acs.analchem.4c05941
Eliise Tammekivi, Karine Faure
In this study, we present the formation of artifacts from simple phenolic compounds and derivatives in SFC-UV-MS analysis. These ions were detected only when the UV detector was turned on, demonstrating that UV light is necessary for their formation. Based on high-resolution mass spectrometry (HRMS) analysis of 21 standards in negative electrospray ionization mode, the artifacts were annotated as ions where CO2 or NO2 had been added to the molecular ion or to an ion that had lost a functional group. In approximately half of the cases, the MS signal of the artifact was higher than that of the molecular ion. Although the formation of artifacts can complicate nontarget analysis as the detected molecular ion does not match with the analyzed standard, we demonstrated that the phenomenon can aid with the structural identification of isomers due to the formation of specific ions. In addition, the overall MS signal increased when the UV was turned on, which can help with the detection of low-abundance compounds, and one compound─ anisole─ was detected only thanks to the artifact. Thus, the aim of this article is to make researchers aware of the UV effect in SFC-UV-MS analysis together with the advantages and disadvantages of artifact formation.
{"title":"Formation of Artifacts from Simple Phenolic Compounds in SFC-UV-(HR)MS","authors":"Eliise Tammekivi, Karine Faure","doi":"10.1021/acs.analchem.4c05941","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05941","url":null,"abstract":"In this study, we present the formation of artifacts from simple phenolic compounds and derivatives in SFC-UV-MS analysis. These ions were detected only when the UV detector was turned on, demonstrating that UV light is necessary for their formation. Based on high-resolution mass spectrometry (HRMS) analysis of 21 standards in negative electrospray ionization mode, the artifacts were annotated as ions where CO<sub>2</sub> or NO<sub>2</sub> had been added to the molecular ion or to an ion that had lost a functional group. In approximately half of the cases, the MS signal of the artifact was higher than that of the molecular ion. Although the formation of artifacts can complicate nontarget analysis as the detected molecular ion does not match with the analyzed standard, we demonstrated that the phenomenon can aid with the structural identification of isomers due to the formation of specific ions. In addition, the overall MS signal increased when the UV was turned on, which can help with the detection of low-abundance compounds, and one compound─ anisole─ was detected only thanks to the artifact. Thus, the aim of this article is to make researchers aware of the UV effect in SFC-UV-MS analysis together with the advantages and disadvantages of artifact formation.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"12 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143443870","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-19DOI: 10.1021/acs.analchem.4c05859
Cailum M. K. Stienstra, Emir Nazdrajić, W. Scott Hopkins
Liquid chromatography (LC) is a cornerstone of analytical separations, but comparing the retention times (RTs) across different LC methods is challenging because of variations in experimental parameters such as column type and solvent gradient. Nevertheless, RTs are powerful metrics in tandem mass spectrometry (MS2) that can reduce false positive rates for metabolite annotation, differentiate isobaric species, and improve peptide identification. Here, we present Graphormer-RT, a novel graph transformer that performs the first single-model method-independent prediction of RTs. We use the RepoRT data set, which contains 142,688 reverse phase (RP) RTs (from 191 methods) and 4,373 HILIC RTs (from 49 methods). Our best RP model (trained and tested on 191 methods) achieved a test set mean average error (MAE) of 29.3 ± 0.6 s, comparable performance to the state-of-the-art model which was only trained on a single LC method. Our best-performing HILIC model achieved a test MAE = 42.4 ± 2.9 s. We expect that Graphormer-RT can be used as an LC “foundation model”, where transfer learning can reduce the amount of training data needed for highly accurate “specialist” models applied to method-specific RP and HILIC tasks. These frameworks could enable the machine optimization of automated LC workflows, improved filtration of candidate structures using predicted RTs, and the in silico annotation of unknown analytes in LC-MS2 measurements.
{"title":"From Reverse Phase Chromatography to HILIC: Graph Transformers Power Method-Independent Machine Learning of Retention Times","authors":"Cailum M. K. Stienstra, Emir Nazdrajić, W. Scott Hopkins","doi":"10.1021/acs.analchem.4c05859","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05859","url":null,"abstract":"Liquid chromatography (LC) is a cornerstone of analytical separations, but comparing the retention times (RTs) across different LC methods is challenging because of variations in experimental parameters such as column type and solvent gradient. Nevertheless, RTs are powerful metrics in tandem mass spectrometry (MS<sup>2</sup>) that can reduce false positive rates for metabolite annotation, differentiate isobaric species, and improve peptide identification. Here, we present Graphormer-RT, a novel graph transformer that performs the first single-model method-independent prediction of RTs. We use the RepoRT data set, which contains 142,688 reverse phase (RP) RTs (from 191 methods) and 4,373 HILIC RTs (from 49 methods). Our best RP model (trained and tested on 191 methods) achieved a test set mean average error (MAE) of 29.3 ± 0.6 s, comparable performance to the state-of-the-art model which was only trained on a single LC method. Our best-performing HILIC model achieved a test MAE = 42.4 ± 2.9 s. We expect that Graphormer-RT can be used as an LC “foundation model”, where transfer learning can reduce the amount of training data needed for highly accurate “specialist” models applied to method-specific RP and HILIC tasks. These frameworks could enable the machine optimization of automated LC workflows, improved filtration of candidate structures using predicted RTs, and the <i>in silico</i> annotation of unknown analytes in LC-MS<sup>2</sup> measurements.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"15 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143452157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
RNA granules are liquid–liquid-phase-separated condensates comprising RNA and proteins. Despite growing insights into their biological functions, studies have predominantly relied on biological methodologies lacking adequate chemical tools. Here, we introduce ICP-CHARGINGS, a concept for efficiently identifying chemical probes to characterize RNA granules of interest among nucleic acid-targeting agents. Focusing on mitochondrial RNA granules (MRGs), whose functions remain elusive, we developed a methodology within this framework and identified NATA, a new fluorescent molecule that, following mechanistic studies, was found to bind to the mitoribosome, enabling MRG labeling and recognition. Using NATA to reveal the potential buffering roles of MRGs, we demonstrated a close correlation between MRG maintenance and assembly and cellular survival and proliferation under cold shock and hypoxic stress. Overall, the introduction and implementation of the ICP-CHARGINGS strategy provide a specialized chemical tool for advancing our comprehension of MRG biology and establish a paradigm for elucidating RNA structures within RNA granules that can be targeted by small molecules, paving the way for developing tailored chemical probes for diverse RNA granules in future research.
{"title":"Deciphering Phase-Separated Mitochondrial RNA Granules under Stress Conditions with the Mitoribosome-Targeting Small Molecule","authors":"Gui-Xue Tang, Shu-Tang Zeng, Jian Wang, Jia-Tong Yan, Shuo-Bin Chen, Zhi-Shu Huang, Xiu-Cai Chen, Jia-Heng Tan","doi":"10.1021/acs.analchem.4c05506","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05506","url":null,"abstract":"RNA granules are liquid–liquid-phase-separated condensates comprising RNA and proteins. Despite growing insights into their biological functions, studies have predominantly relied on biological methodologies lacking adequate chemical tools. Here, we introduce ICP-CHARGINGS, a concept for efficiently identifying chemical probes to characterize RNA granules of interest among nucleic acid-targeting agents. Focusing on mitochondrial RNA granules (MRGs), whose functions remain elusive, we developed a methodology within this framework and identified <b>NATA</b>, a new fluorescent molecule that, following mechanistic studies, was found to bind to the mitoribosome, enabling MRG labeling and recognition. Using <b>NATA</b> to reveal the potential buffering roles of MRGs, we demonstrated a close correlation between MRG maintenance and assembly and cellular survival and proliferation under cold shock and hypoxic stress. Overall, the introduction and implementation of the ICP-CHARGINGS strategy provide a specialized chemical tool for advancing our comprehension of MRG biology and establish a paradigm for elucidating RNA structures within RNA granules that can be targeted by small molecules, paving the way for developing tailored chemical probes for diverse RNA granules in future research.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"181 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143443869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-19DOI: 10.1021/acs.analchem.4c05964
Jon Henrik Both, Anastasiya Beliakouskaya, Karl-Michael Weitzel
The quantification of molar fractions and enantiomeric excess has been demonstrated in mixtures of d- and l-tryptophan and d- and l-phenylalanine, respectively, avoiding derivatization of the analyte with additional reagents or separation steps. The technique is based on electrospray ionization (ESI), which allows the generation of anions of nonvolatile compounds such as amino acids or large biomolecules. Electrons are photodetached from these anions. The distribution of forward and backward scattered photoelectrons is analyzed, leading to photoelectron circular dichroism (PECD), the observable of interest. The quantification of the concept is proven by blind measurements analyzing mixtures of unknown composition. The quantification of enantiomeric excess (ee) values is not only possible for signals originating from the molecular anion but also for the molecular dimer anion. The ESI-PECD technique is known to be applicable to large chemical entities of several thousand Daltons.
{"title":"Determination of the Molar Fraction and Enantiomeric Excess of Electrosprayed Amino Acid Anions Employing Photoelectron Circular Dichroism","authors":"Jon Henrik Both, Anastasiya Beliakouskaya, Karl-Michael Weitzel","doi":"10.1021/acs.analchem.4c05964","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05964","url":null,"abstract":"The quantification of molar fractions and enantiomeric excess has been demonstrated in mixtures of <span>d-</span> and <span>l</span>-tryptophan and <span>d-</span> and <span>l-</span>phenylalanine, respectively, avoiding derivatization of the analyte with additional reagents or separation steps. The technique is based on electrospray ionization (ESI), which allows the generation of anions of nonvolatile compounds such as amino acids or large biomolecules. Electrons are photodetached from these anions. The distribution of forward and backward scattered photoelectrons is analyzed, leading to photoelectron circular dichroism (PECD), the observable of interest. The quantification of the concept is proven by blind measurements analyzing mixtures of unknown composition. The quantification of enantiomeric excess (ee) values is not only possible for signals originating from the molecular anion but also for the molecular dimer anion. The ESI-PECD technique is known to be applicable to large chemical entities of several thousand Daltons.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"35 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143443872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-19DOI: 10.1021/acs.analchem.4c06426
Shuo Wang, Bin Hu, Han Wang, Xingyi Qi, Zhuo Mi, Ping Su, Huaiyu Zhang, Jianfeng Zhou, Jianchun Chen, Baorong Chen, Jiayi Song, Yi Yang
Angiotensin I, II, and III (Ang I, II, and III) and aldosterone (Aldo) play an important role in primary aldosteronism (PA) screening according to the study of the blood pressure regulation mechanism. However, Ang I, Ang II, Ang III, and Aldo are present in human plasma at low concentrations and have different polarities, which make it rather challenging for current detection methods to simultaneously detect four analytes in complex blood samples. In this study, a new magnetic covalent organic framework (COF) was synthesized for the enrichment of Ang I, II, and III and Aldo in human plasma, and a new method for the simultaneous detection of four analytes was developed based on the magnetic COF and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The experimental results demonstrated that the adsorption kinetics of the material conformed to a pseudo-second-order model, and the enrichment mechanisms were π-π stacking, electrostatic, and hydrogen bonding interactions. Under the optimized conditions, the established method has satisfactory linear ranges (Ang I: 100–25,000 pg/mL, Ang II: 2–500 pg/mL, Ang III: 3–750 pg/mL, and Aldo: 20–5000 pg/mL), a low limit of detection (0.8–5 pg/mL), high recoveries (93.0–111.3%), and multiple recycling, which were superior to those reported studies. Meanwhile, the results of testing 20 clinical samples indicated that Ang I, Ang II, Ang III, and Aldo were effective and could be used as new biomarkers for PA screening, which proved the feasibility of enriching the four targets in real blood samples. The prepared magnetic COF in this experiment provided a reference for the material design to simultaneous enrichment of Ang I, Ang II, Ang III, and Aldo, and the developed new method based on the magnetic COF and LC-MS/MS provided a new detection idea for PA screening, which greatly promoted the development of PA disease diagnosis and was expected to be used in further clinical research.
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