{"title":"Online LC-MS/MS Analysis for Profiling Peptide Hormone Secretion Dynamics from Islets of Langerhans","authors":"Joshua J. Davis, James Thornham, Michael G. Roper","doi":"10.1021/acs.analchem.4c06643","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06643","url":null,"abstract":"Multiple peptide hormones are secreted from islets of Langerhans to maintain blood glucose homeostasis. Defects in the amount and patterns of hormone secretion can lead to metabolic disorders, such as diabetes. To understand the relationships between the peptides, analytical methods that can quantify multiple hormones in short time increments are required. To this end, an automated and online system was developed for sampling perfusate from ∼30 human islets held on a glass microfluidic device with sequential liquid chromatography (LC)-MS/MS runs every 2 min to resolve secretion dynamics. Islet perfusate was mixed with an isotopically labeled internal standard and loaded into a 2 μL sample loop which was injected for 0.1 min every 1.9 min onto a 2.1 mm × 30 mm (I.D. × length) C18 column held at 70 °C. Online detection of insulin, C-peptide, glucagon, and somatostatin levels was performed using a triple quadrupole mass spectrometer. Optimization of separation conditions using linear solvent strength theory enabled rapid separation of the four peptides. Calibration curves were linear from 0.5 to 50 nM with an RSD for all analytes between 3–15% and <3% RSD for all retention times. Results showed secretion dynamics such as first-phase insulin release and negatively correlated release of glucagon and insulin. This simple LC-MS method that used a single 6-port valve with a single sample loop is expected to be useful for examining secretion of other biologically relevant molecules from islets and could be applied to other biological systems for rapid and automated sampling to investigate cellular communication.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"12 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143385675","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}
Analytical ChemistryPub Date : 2025-02-11Epub Date: 2025-01-31DOI: 10.1021/acs.analchem.4c05736
Lijia Xu, Yanqi Feng, Ao Feng, Yuping Yang, Yanjun Chen, Bo Liu, Ning Yang, Wei Ma, Yong He, Zhijun Wu, Yuchao Wang, Yongpeng Zhao
{"title":"Study on Rapid Quantitative Detection of Soil MPs Based on Terahertz Time-Domain Spectroscopy.","authors":"Lijia Xu, Yanqi Feng, Ao Feng, Yuping Yang, Yanjun Chen, Bo Liu, Ning Yang, Wei Ma, Yong He, Zhijun Wu, Yuchao Wang, Yongpeng Zhao","doi":"10.1021/acs.analchem.4c05736","DOIUrl":"10.1021/acs.analchem.4c05736","url":null,"abstract":"<p><p>The presence of microplastics (MPs) in agricultural soils substantially affects the growth, reproduction, feeding, survival, and immunity levels of soil biota. Therefore, it is crucial to investigate fast, effective, and accurate techniques for the detection of soil MPs. This work explores the integration of terahertz time-domain spectroscopy (THz-TDS) techniques with machine learning algorithms to develop a method for the classification and detection of MPs. First, THz spectral image data were preprocessed using moving average (MA). Subsequently, three classification models were developed, including random forest (RF), linear discriminant analysis, and support vector machine (SVM). Notably, the SVM model had an F1 score of 0.9817, demonstrating its ability to rapidly classify MPs in soil samples. Three regression models, namely, principal component regression (PCR), RF, and least squares support vector machine (LSSVM), were developed for the detection of three MPs polymers in agricultural soils. Six feature extraction methods were used to extract the relevant parts of the data containing key information. The results of the study showed that the regression accuracies of PCR, RF, and LSSVM were greater than 83%. Among them, the RF had the highest overall regression accuracy. Notably, PE-UVE-RF had the best performance with <i>R</i><sub>c</sub><sup>2</sup>, <i>R</i><sub>p</sub><sup>2</sup>, root mean square error of calibration, and root mean square error of prediction values of 0.9974, 0.9916, 0.1595, and 0.2680, respectively. Furthermore, this model gets a better performance by hypothesis testing and predicting real samples.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":" ","pages":"2952-2962"},"PeriodicalIF":6.7,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062137","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}
{"title":"A Bacteria-Responsive Multifunctional Nanohydrogel for Recognition of Bacterial Infections and Activable Four-in-One Antibacterial Therapy.","authors":"Haochen Li, Ziqian Xu, Haocong Sun, Yangyang Cai, Shangmei Zhou, Peng Zhang, Ke Zheng, Caifeng Ding","doi":"10.1021/acs.analchem.4c06251","DOIUrl":"10.1021/acs.analchem.4c06251","url":null,"abstract":"<p><p>Bacterial infections have long been a formidable challenge in global public health, further compounded by the emergence of drug-resistant bacteria resulting from the overuse and misuse of antibiotics. Intelligent antibacterial strategies are garnering escalating attention and concern due to their ability to accurately recognize bacterial infections, efficiently eliminate pathogens, and timely monitor infection end points in order to mitigate the adverse effects of excessive treatment on normal tissues. Hence, in this study, we developed a multifunctional antibacterial nanohydrogel that exhibited bacteria-triggered fluorescence activity, serving as a fluorescent indicator for bacterial infections. Moreover, the bacteria can induce the release of Fe<sup>3+</sup>, photosensitizers, and antibiotics within the nanohydrogel, thereby exerting synergistic antibacterial effects through chemodynamic and photodynamic treatment, glutathione depletion, and antibiotics. Consequently, the nanohydrogel demonstrated remarkable efficacy in eradicating bacteria within wounds while significantly enhancing wound healing. The construction strategy and design principles of the antibacterial nanohydrogel broaden the horizons of clinical photodynamic antibacterial therapy, offering a novel perspective for the advancement of integrated theranostic approaches against bacterial infections.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":" ","pages":"3074-3082"},"PeriodicalIF":6.7,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062286","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}
{"title":"Ferrous Tungstate Nanomaterials with Excellent Enzyme-Mimicking Activity to Enhance Lateral Flow Immunoassay Sensitivity.","authors":"Xianqing Tang, Hua Zhang, Jinghuang Chen, Donghui Wu, Yu Wang, Jian Sun, Zhenxin Wang, Xiurong Yang","doi":"10.1021/acs.analchem.4c04761","DOIUrl":"10.1021/acs.analchem.4c04761","url":null,"abstract":"<p><p>Lateral flow immunochromatography (LFIA) with gold nanoparticles (AuNPs) is widely used in the biomedical field as a rapid and simple in vitro detection technique. However, the conventional AuNP-LFIA has limitations in sensitivity and detection range. In this study, nonprecious metal iron-based bimetallic FeWO<sub>4</sub> nanomaterials with convenient and excellent enzyme-mimetic catalytic activities were synthesized by a one-pot hydrothermal method. Here, FeWO<sub>4</sub> nanomaterials were combined with C-reactive protein (CRP) detection antibodies to form a novel signal-enhancing probe for the analysis of CRP. The probe further achieves significant signal amplification by catalyzing the oxidation reaction of 3-amino-9-ethylcarbazole in the LFIA assay, thereby improving the sensitivity and accuracy of the assay. The application of FeWO<sub>4</sub>-based LFIA improved the limit of detection of CRP to 19.38 ng/mL after catalytic amplification, which is approximately 30-fold lower than that of the conventional AuNP-LFIA method. In addition, the method demonstrated good stability and reproducibility, providing a promising and prospective strategy for the early diagnosis of inflammatory diseases.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":" ","pages":"2714-2723"},"PeriodicalIF":6.7,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062305","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}
Analytical ChemistryPub Date : 2025-02-11Epub Date: 2025-01-31DOI: 10.1021/acs.analchem.4c04355
Damien Olivier-Jimenez, Rico J E Derks, Oscar Harari, Carlos Cruchaga, Muhammad Ali, Alessandro Ori, Domenico Di Fraia, Birol Cabukusta, Andy Henrie, Martin Giera, Yassene Mohammed
{"title":"iSODA: A Comprehensive Tool for Integrative Omics Data Analysis in Single- and Multi-Omics Experiments.","authors":"Damien Olivier-Jimenez, Rico J E Derks, Oscar Harari, Carlos Cruchaga, Muhammad Ali, Alessandro Ori, Domenico Di Fraia, Birol Cabukusta, Andy Henrie, Martin Giera, Yassene Mohammed","doi":"10.1021/acs.analchem.4c04355","DOIUrl":"10.1021/acs.analchem.4c04355","url":null,"abstract":"<p><p>Thanks to the plummeting costs of continuously evolving omics analytical platforms, research centers collect multiomics data more routinely. They are, however, confronted with the lack of a versatile software solution to harmoniously analyze single-omics and interpret multiomics data. We have developed iSODA, a web-based application for the analysis of single- and multiomics data. The tool emphasizes intuitive interactive visualizations designed for user-driven data exploration. Researchers can access a variety of functions ranging from simple visualization like volcano plots and PCA to advanced functional analyses like enrichment analysis and lipid saturation analysis. For integrated multiomics, iSODA incorporates multi-omics factor analysis and similarity network fusion. The ability to adapt the data on-the-fly allows for tasks, such as the removal of outlier samples or failed features, imputation, or normalization. All results are presented through interactive plots, the modular design supports extensions, and tooltips and tutorials provide comprehensive guidance for users. iSODA is accessible under http://isoda.online/.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":" ","pages":"2689-2697"},"PeriodicalIF":6.7,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062327","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}
{"title":"Consolidated Microscale Interferon-γ Release Assay with Tip Optofluidic Immunoassay for Dynamic Parallel Diagnosis of Tuberculosis Infection.","authors":"Binmao Zhang, Yuzhong Xu, Zhen Huang, Ruihan Li, Tianen Zhu, Shangyan Liang, Hao Huang, Siyan Zhong, Hui Yang, Xudong Fan, Xiaotian Tan, Yujuan Chai","doi":"10.1021/acs.analchem.4c05390","DOIUrl":"10.1021/acs.analchem.4c05390","url":null,"abstract":"<p><p>Interferon-γ release assay (IGRA) is one of the most important diagnostic tools for tuberculosis (TB) infection. Despite its high accuracy, conventional IGRA has several drawbacks, including complicated procedures, large blood volume requirements, lengthy incubation times, and difficulties in parallel testing. Efforts have been made to develop miniaturized and highly sensitive biosensors for interferon-γ or to evaluate the specific immune response through microfluidic platforms. However, the need for sophisticated consumables and equipment, as well as the partial experimental design, has limited the application of these advanced techniques in TB diagnosis and disease control. Here, we report the development of a tip optofluidic immunoassay (TOI)-based consolidated microscale IGRA (CM-IGRA) for the dynamic and parallel evaluation of TB infection, refining both the blood incubation and interferon-γ quantification processes. The TOI system comprises 12 microfluidic immuno-reactors and a portable chemiluminescent imaging station, capable of quantifying interferon-γ with high sensitivity (8.00 pg/mL in plasma) and a wide detection range (∼10<sup>4</sup>). The results generated with CM-IGRA achieved 98.39% agreement with the standard IGRA while reducing blood sample consumption to 50 μL per assay (20-fold reduction) and significantly shortening the incubation time from 20 to 10 h. This diagnostic method simplifies operations and improves efficiency for the parallel assays required in IGRA, providing a promising solution for TB screening in patients for whom current methods are inconvenient, such as children and older adults.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":" ","pages":"2863-2872"},"PeriodicalIF":6.7,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143070727","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}
Analytical ChemistryPub Date : 2025-02-11Epub Date: 2025-01-31DOI: 10.1021/acs.analchem.4c06066
Di Huang, Yichen He, Chutian Xu, Peijie Shen, Min Li, Mengjun Fang, Zhinan Xu, Xiangming Fang
{"title":"DNAzyme-Triggered Equilibrium Transfer with Self-Activated CRISPR-Cas12a Biosensor Enables One-Pot Diagnosis of Nucleic Acids.","authors":"Di Huang, Yichen He, Chutian Xu, Peijie Shen, Min Li, Mengjun Fang, Zhinan Xu, Xiangming Fang","doi":"10.1021/acs.analchem.4c06066","DOIUrl":"10.1021/acs.analchem.4c06066","url":null,"abstract":"<p><p>Integrating recombinase-polymerase amplification (RPA) with CRISPR-Cas12a holds significant potential to simplify and improve nucleic acid diagnostic procedures. However, current strategies face limitations, such as complexity, reduced efficiency, and potential compromises in Cas12a activity. In response, we developed a <u>D</u>NAzyme-triggered <u>e</u>quilibrium transfer with a <u>s</u>elf-activated <u>CRI</u>SPR-Cas12a <u>b</u>ios<u>e</u>nso<u>r</u> (DESCRIBER) for integrated nucleic acid detection. This platform features varying balance points to minimize interference between RPA and Cas12a in one pot and maximize their activity at different stages. Initially, the reaction focused on RPA, while Cas12a was silenced by circular-crRNA (C-crRNA). Then, DNAzyme, the activator, was generated during the RPA process, which linearizes C-crRNA to activate Cas12a and transfer the equilibrium toward signal readout. Meanwhile, activated Cas12a can further linearize C-crRNA to promote self-activation and accelerate equilibrium transfer. According to this principle, highly sensitive detection of the HIV-1 genome, as low as 500 CPs/mL, was achieved within 1 h while maintaining universality in detecting common subtypes and specificity against opportunistic infectious pathogens. Compared with qRT-PCR, it also exhibited good accuracy in detecting 35 spiked samples. Overall, we believe that the proposed strategy will enhance existing CRISPR systems to promote their practical applications in clinical diagnosis.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":" ","pages":"3026-3035"},"PeriodicalIF":6.7,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143070729","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}
{"title":"Ultrasensitive COFs Functionalized Electrochemical Biosensor for DNA Methyltransferase Activity Detection by DNA Walking and Rolled Circular Strand Displacement Amplification","authors":"Jialin Zhang, Biyao Mao, Shuoshuo Cheng, Kangqiang Lu, Herui Wen, Jiali Ren","doi":"10.1021/acs.analchem.4c06572","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06572","url":null,"abstract":"Assessing the activity of DNA methyltransferases (MTases) and screening for methyltransferase inhibitors not only allow for a deep exploration of the role of methylation regulation in disease initiation and progression but also provide an important experimental and clinical basis for the diagnosis and treatment of diseases. Herein, a new COFs functionalized electrochemical biosensor has been developed to detect DNA adenine methylation (Dam) MTase activity with high sensitivity and rapidity by taking advantage of the DNA walker and rolled circular strand displacement amplification (RC-SDA) reaction. Specifically, hairpin probe H1 was methylated by Dam MTase, followed by methylation site-specific cleavage of DpnI enzyme to generate the S5 probe. The padlock probes with two nucleic acid endonuclease sites were introduced to trigger the RC-SDA reaction under the action of the primer, releasing a large number of single-stranded S6 probes. The released probe worked synergistically with the Exo III enzyme to trigger DNA walking, which exposed the binding sites to enable the Au-COF-MB signal probes to bind effectively to the electrode surface, and the electrochemical signals were thus generated. The result showed that the designed electrochemical biosensor demonstrated excellent sensitivity and specificity in detecting the activity of Dam MTase, with the detection limit (LOD) of 6.85 × 10<sup>–5</sup> U/mL. This method provides support for the development of new treatment strategies for methylation related diseases.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"208 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143385674","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}
Analytical ChemistryPub Date : 2025-02-11Epub Date: 2025-01-31DOI: 10.1021/acs.analchem.4c06015
Min Deng, Peipei Wang, Zibo Zhai, Ying Liu, Dan Cheng, Longwei He, Songjiao Li
{"title":"A Triple-Responsive and Dual-NIR Emissive Fluorescence Probe for Precise Cancer Imaging and Therapy by Activating Pyroptosis Pathway.","authors":"Min Deng, Peipei Wang, Zibo Zhai, Ying Liu, Dan Cheng, Longwei He, Songjiao Li","doi":"10.1021/acs.analchem.4c06015","DOIUrl":"10.1021/acs.analchem.4c06015","url":null,"abstract":"<p><p>Revealing changes in the tumor microenvironment is crucial for understanding cancer and developing sensitive methods for precise cancer imaging and diagnosis. Intracellular hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and microenvironmental factors (e.g., viscosity and polarity) are closely linked to various physiological and pathological processes, making them potential biomarkers for cancer. However, a triple-response theranostic probe for precise tumor imaging and therapy has not yet been achieved due to the lack of effective tools. Herein, we present a mitochondria-targeting near-infrared (NIR) fluorescent probe, <b>VPH-5DF</b>, capable of simultaneously monitoring H<sub>2</sub>O<sub>2</sub>, viscosity, and polarity through dual NIR channels. The probe specifically detects H<sub>2</sub>O<sub>2</sub> via NIR emission (λ<sub>em</sub> = 650 nm) and shows high sensitivity to microenvironmental viscosity/polarity in the deep NIR channel (λ<sub>em</sub> ≈ 750 nm). Furthermore, the probe not only monitors mitochondrial polarity, viscosity, and fluctuations in endogenous/exogenous H<sub>2</sub>O<sub>2</sub> levels but also distinguishes cancer cells from normal cells through multiple parameters. Additionally, VPH-5DF can be employed to monitor alterations in H<sub>2</sub>O<sub>2</sub> levels, as well as changes in viscosity and polarity, during drug-induced pyroptosis in living cells. After treatment with VPH-5DF, chemotherapy-induced oxidative damage to the mitochondria in tumor cells activated the pyroptosis pathway, leading to a robust antitumor response, as evidenced in xenograft tumor models. Thus, this triple-response theranostic prodrug offers a new platform for precise <i>in vivo</i> cancer diagnosis and anticancer chemotherapy.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":" ","pages":"2998-3008"},"PeriodicalIF":6.7,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062292","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}
Analytical ChemistryPub Date : 2025-02-11Epub Date: 2025-01-31DOI: 10.1021/acs.analchem.4c03323
Rania Benazza, Léa Letissier, Greg Papadakos, Jen Thom, Helene Diemer, Graham Cotton, Sarah Cianférani, Oscar Hernandez-Alba
{"title":"Development of Top-Down Mass Spectrometry Strategies in the Chromatographic Time Scale (LC-TD-MS) for the Extended Characterization of an Anti-EGFR Single-Domain Antibody-Drug Conjugate in Both Reduced and Nonreduced Forms.","authors":"Rania Benazza, Léa Letissier, Greg Papadakos, Jen Thom, Helene Diemer, Graham Cotton, Sarah Cianférani, Oscar Hernandez-Alba","doi":"10.1021/acs.analchem.4c03323","DOIUrl":"10.1021/acs.analchem.4c03323","url":null,"abstract":"<p><p>Even though mAbs have attracted the biggest interest in the development of therapeutic proteins, next-generation therapeutics such as single-domain antibodies (sdAb) are propelling increasing attention as new alternatives with appealing applications in different clinical areas. These constructs are small therapeutic proteins formed by a variable domain of the heavy chain of an antibody with multiple therapeutic and production benefits compared with their mAb counterparts. These proteins can be subjected to different bioconjugation processes to form single-domain antibody-drug conjugates (sdADCs) and hence increase their therapeutic potency, and akin to other therapeutic proteins, nanobodies and related products require dedicated analytical strategies to fully characterize their primary structure prior to their release to the market. In this study, we report for the first time the extensive sequence characterization of a conjugated anti-EGFR 14 kDa sdADC by using state-of-the-art top-down mass spectrometry strategies in combination with liquid chromatography (LC-TD-MS). Mass analysis revealed a highly homogeneous sample with one conjugated molecule. Subsequently, the reduced sdADC was submitted to different fragmentation techniques, namely, higher-energy collisional dissociation, electron-transfer dissociation, and electron-transfer higher-energy collision dissociation, allowing to unambiguously assess the conjugation site with 24 diagnostic fragment ions and 85% of global sequence coverage. The sequence coverage of the nonreduced protein was significantly lower (around 16%); however, the analysis of the fragmentation spectra corroborated the presence of the intramolecular disulfide bridge along with the localization of the conjugation site. Altogether, our results pinpoint the difficulties and challenges associated with the fragmentation of sdAb-derived formats in the LC time scale due to their remarkable stability as a consequence of the intramolecular disulfide bridge. However, the use of complementary activation techniques along with the identification of specific ion fragments allows an improved sequence coverage, the characterization of the intramolecular disulfide bond, and the unambiguous localization of the conjugation site.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":" ","pages":"2639-2647"},"PeriodicalIF":6.7,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143070728","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}