Analytical biochemistry最新文献

{"title":"Impact of anticoagulants and centrifugal forces on plasma neutrophil extracellular traps assay outcomes","authors":"Yang Zhang ,&nbsp;Shengqiang Pei ,&nbsp;Dabuxilite Bayartaikishigtai ,&nbsp;Zixin Chen ,&nbsp;Hongtao Zhang ,&nbsp;Wei Chen","doi":"10.1016/j.ab.2025.116037","DOIUrl":"10.1016/j.ab.2025.116037","url":null,"abstract":"<div><h3>Background</h3><div>Neutrophil extracellular traps (NETs) could help prevent and treat thrombosis-related diseases. Detecting NETs primarily relies on plasma; however, the impact of anticoagulants and plasma centrifugation on these results remains unclear.</div></div><div><h3>Objectives</h3><div>This study aimed to identify the most effective anticoagulant and centrifugation methods for detecting NETs in plasma.</div></div><div><h3>Methods</h3><div>Plasma samples were collected from sepsis rats using sodium citrate and EDTA as anticoagulants. These samples were subjected to various centrifugal forces, durations, and temperatures to determine the optimal method for NETs analysis. Similarly, plasma samples from infected patients were collected and analyzed using different centrifugation techniques to identify the optimal method. Gene expression analysis in endothelial cells stimulated by these plasma samples was conducted to compare the endothelial responses to NETs levels.</div></div><div><h3>Results</h3><div>In rats, EDTA-plasma exhibited increased levels of myeloperoxidase (MPO)-DNA, while dsDNA levels remained unchanged, with both concentrations being higher at 400 g. Similarly, EDTA and a centrifugal force of 400 g increased MPO-DNA levels without affecting dsDNA levels in patients. Plasma isolated using a centrifugal force of 400 g enhanced adhesion, inflammation, and chemotaxis gene expression in endothelial cells, demonstrating that low centrifugal force is effective for NETs detection.</div></div><div><h3>Conclusion</h3><div>For an accurate NETs assay, it is recommended to use EDTA anticoagulant and prepare plasma using a low centrifugal force, such as 400g.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116037"},"PeriodicalIF":2.5,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145848761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Oxidative interference in immunoturbidimetric assays for the quantification of immunoglobulins and complement proteins","authors":"Bruna Garlet Rossato ,&nbsp;Rafael Noal Moresco","doi":"10.1016/j.ab.2026.116050","DOIUrl":"10.1016/j.ab.2026.116050","url":null,"abstract":"<div><div>Immunoturbidimetric assays are widely used to quantify immunoglobulins and complement proteins but are susceptible to interference. This study evaluated the effects of oxidative agents, including the Fenton reaction and hypochlorous acid (HOCl), on these assays. Serum pools were oxidized <em>in vitro</em>, and IgA, IgG, IgM, C3, and C4 were measured. Oxidative conditions increased measured concentrations of IgA, IgM, C3, and C4, whereas IgG was unaffected by HOCl. Increased oxidative stress index values and reduced thiol levels confirmed oxidative modification, indicating potential impairment of assay accuracy under oxidative stress.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116050"},"PeriodicalIF":2.5,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145951142","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multi-disciplinary combination to explore the laxation mechanism of Rhei Radix et Rhizoma and Magnoliae Officinalis Cortex","authors":"Meiyu Wan,&nbsp;Yu Wang,&nbsp;Meijuan Liu,&nbsp;Wenwen Zhou,&nbsp;Xiaoxiao Zhang,&nbsp;Mingyang Wang,&nbsp;Shu Jiang,&nbsp;Erxin Shang,&nbsp;Jinao Duan","doi":"10.1016/j.ab.2025.116030","DOIUrl":"10.1016/j.ab.2025.116030","url":null,"abstract":"<div><h3>Background</h3><div>The combination of Rhei Radix et Rhizoma (RR) and Magnoliae Officinalis Cortex (MO) has been used to treat constipation for thousands of years. However, the synergistic mechanism of two herbs to exert the laxative effects is still unclear.</div></div><div><h3>Materials and methods</h3><div>In this study, the constipation rat model was successfully induced by loperamide hydrochloride, which was applied to systematically and comprehensively evaluate the effect of RR and MO in ameliorating the constipation. Additionally, network pharmacology, molecular docking and UPLC-Q-TOF/MS-based metabolomics were integrated to uncover the potential mechanism of the compatibility of RR and MO. And MetaboAnalyst and MetaScape jointly analyze the metabolic pathways involved in network pharmacology targets and metabolomics metabolites, selecting key metabolic pathways.</div></div><div><h3>Results</h3><div>In terms of physiological and biochemical level, the laxative effect of RR and MO was significantly better than that of single herb. The results of network pharmacology and molecular docking showed that RR and MO could improve the constipation through multi-components, multi-targets and multi-pathways. Moreover, metabolomics indicated that the disrupted metabolic profile in plasma of model rats was markedly reversed by the combination treatment of RR and MO. Additionally, after a comprehensive analysis of 9 metabolites and 124 targets, arachidonic acid was selected as the most critical pathway. The results showed that the herb pair could relieve constipation through arachidonic acid pathway and inhibited the levels of PTGS2 and PLA2G2A protein in this pathway.</div></div><div><h3>Conclusion</h3><div>This study revealed the synergistic effect of the combination of RR and MO in improving the constipation from the perspective of bioinformatics and metabolomics. Furthermore, the integrated method of multi-omics could contribute to exploring the potential compatibility mechanism of traditional Chinese medicines.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"710 ","pages":"Article 116030"},"PeriodicalIF":2.5,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145735328","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Trypsin digestion to prevent acid induced precipitation of metalloproteins for accurate ICP-MS analysis: Nitrogenase case study","authors":"Jessica K. Bilyj,&nbsp;Christina M. Gregg,&nbsp;Trevor D. Rapson","doi":"10.1016/j.ab.2025.116035","DOIUrl":"10.1016/j.ab.2025.116035","url":null,"abstract":"<div><div>Accurate determination of metallation states in metalloenzymes is essential for correlating metal content with enzymatic function. Inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectroscopy (ICP-OES) are among the most sensitive and practical techniques for this purpose. However, complete digestion of protein samples is critical for accurate results, and the use of acid often leads to protein precipitation before the metals are fully released into solution.</div><div>Here, we present a case study using the nitrogenase protein NifDK, which contains both Fe and Mo, to demonstrate that trypsin digestion can prevent acid-induced precipitation. A 1:1 (w/w) ratio of protein to trypsin in Tris buffer at 37 °C overnight results in complete digestion of NifDK. Upon acid addition, a homogeneous solution is obtained without precipitation. This approach yields highly reproducible ICP-MS data.</div><div>Further improvements include performing protein quantification on the exact vial used for ICP-MS analysis, applying a drift correction to the ICP-MS data, and using nitric acid with 0.1 % (v/v) HF for accurate molybdenum quantification.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"710 ","pages":"Article 116035"},"PeriodicalIF":2.5,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145802967","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The application of machine learning in nuclear magnetic resonance (NMR) peak selection for metabolomics studies","authors":"Moses Mayonu ,&nbsp;Saeedeh Babaee ,&nbsp;Julie Pollak ,&nbsp;Lin Jiang ,&nbsp;Bo Wang","doi":"10.1016/j.ab.2025.116034","DOIUrl":"10.1016/j.ab.2025.116034","url":null,"abstract":"<div><div>Nuclear magnetic resonance (NMR), which is famous for its nondestructive nature and high reliability, is one of the principal analytical platforms in metabolomics<strong>.</strong> NMR metabolomics has been widely used in human and environmental health studies in the past few decades. However, NMR metabolomics data processing remains challenging due to data complexity. Although automated approaches have been explored, their reliability and accuracy are still limited. One of the limitations is the lack of cross-evaluation of the same peak in all samples during raw data processing. In this study, we developed a new approach that applies machine learning models to evaluate the peak quality for all the samples in an NMR metabolomics study. Our new approach combines the automatically selected potential peaks from all samples into a new spectrum for each peak (potential metabolite), which provides an overview of all the samples to ensure the overall data quality for the downstream statistical analysis. The results indicated that two machine learning approaches, Support Vector Machine Discriminant Analysis (SVMDA) and Extreme Gradient Boosting Discriminant Analysis (XGBDA), demonstrated high prediction rates in identifying high-quality peaks. In addition, the raw data conversion resolution was tested to optimize the performance of each machine learning approach, and XGBDA showed better tolerance to data resolution. The results indicated that machine learning approaches, such as SVMDA and XGBDA, can be used to identify high-quality peaks generated through automated peak picking, ensuring data quality for metabolomics studies. Our study paves the way for automated data processing in future NMR metabolomics research.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"710 ","pages":"Article 116034"},"PeriodicalIF":2.5,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145800380","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Classification prediction of drug target binding affinity based on the MolrProtTrans model","authors":"Yicun Lin,&nbsp;Yuanfeng Li,&nbsp;Wei Sun,&nbsp;Jian Wang","doi":"10.1016/j.ab.2025.116029","DOIUrl":"10.1016/j.ab.2025.116029","url":null,"abstract":"<div><div>Predicting drug-target interactions is essential for virtual drug screening. While many models predict the binding affinity between small molecules and proteins, they often overemphasize molecular features while overlooking important protein characteristics, leading to biased predictions. Traditional deep learning models, such as transformerCPI, show limited performance in tasks like label inversion on G protein-coupled receptor (GPCR) datasets. To address this, this study proposes an enhanced transformer-based model that integrates both molecular and protein information. By leveraging Molr and ProtTrans networks for feature extraction, and incorporating a transposed attention mechanism with a triple-loss self-supervised learning approach, the model improves prediction accuracy. Experimental results show that the proposed model achieved observed Area Under the Curve (AUC) of 0.81 on the GPCR label-inversion dataset and 0.92 on a human target dataset, which are numerically higher than those reported for TransformerCPI and several baseline methods in our experiments. These observations indicate improved performance in our experimental setting, offering promising prospects for advancing virtual drug screening and drug discovery.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"710 ","pages":"Article 116029"},"PeriodicalIF":2.5,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145695935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimized purification workflow for advanced eEF1A studies: Impact of His-tagging on stability, functionality, and yield","authors":"Pernille Nedergaard Madsen,&nbsp;Pernille Baden Jørgensen,&nbsp;Mille Varisbøl Clausen,&nbsp;Charlotte Rohde Knudsen","doi":"10.1016/j.ab.2025.116017","DOIUrl":"10.1016/j.ab.2025.116017","url":null,"abstract":"<div><div>The eukaryotic elongation factor 1A (eEF1A) is crucial for translation, delivering aminoacyl-tRNAs to the ribosomal A site. While its bacterial counterpart, elongation factor thermo unstable (EF-Tu), has been extensively studied, the canonical and non-canonical roles of eEF1A remain less understood, partly due to the lack of an efficient purification method. This study optimized an affinity chromatography-based protocol for <em>Saccharomyces cerevisiae</em> eEF1A, evaluating the effects of N- and C-terminal His-tagging on stability, functionality, purification, and yield. While N-terminal deletion of four residues impaired ternary complex formation <em>in vitro</em>, His-tagging at the same position did not. However, nano differential scanning fluorimetry (NanoDSF) revealed that an N-terminal His-tag destabilizes eEF1A, whereas a C-terminal His-tag preserves its integrity and enhances yield. Additionally, reducing glycerol concentration from 25 % to 10 % expedited purification without compromising stability or tRNA binding. This optimized C-terminal His-tag protocol provides a streamlined approach for studying the functional and dynamic properties of eEF1A.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"710 ","pages":"Article 116017"},"PeriodicalIF":2.5,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145675946","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of the function of the metabolite repair enzyme Nit1: the story of a collaboration with Arthur Cooper","authors":"Emile Van Schaftingen ,&nbsp;Alessio Peracchi ,&nbsp;Maria Veiga-da-Cunha","doi":"10.1016/j.ab.2025.116032","DOIUrl":"10.1016/j.ab.2025.116032","url":null,"abstract":"<div><div>Nit1 and Nit2 were initially identified in the context of cancer research, as proteins encoded by putative (anti)oncogenes. However, the presence of homologous proteins in bacteria suggested that they might be enzymes with a fundamental metabolic function. Our group, while interacting with Arthur Cooper and his collaborators, contributed to uncovering these roles: Nit2 was identified in 2009 as an ω-amidase, the enzyme that hydrolyses the ‘ω’ amide of α-ketoglutaramate and α-ketosuccinamate, the ‘deaminated’ derivatives of glutamine and asparagine produced in some irreversible transamination reactions. Later, in 2017, we showed that Nit1 functions as a metabolite-repair enzyme. Specifically, Nit1 efficiently hydrolyzes deaminated gluthathione (dGSH), a non-functional byproduct generated by a side activity of various classical transaminases. This repair function prevents the accumulation of the useless metabolite dGSH. The physiological significance of Nit1 is underscored by recent discoveries linking its deficiency in humans to a neurological disorder.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"710 ","pages":"Article 116032"},"PeriodicalIF":2.5,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145751493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Direct detection of a target nucleic acid on a surface by spraying DNA-nano-tweezers-based biosensing molecules","authors":"Hisakage Funabashi,&nbsp;Hiroya Hatano,&nbsp;Hirotaka Nakamura,&nbsp;Reiji Shigematsu,&nbsp;Akio Kuroda","doi":"10.1016/j.ab.2025.116003","DOIUrl":"10.1016/j.ab.2025.116003","url":null,"abstract":"<div><div>We propose a new concept to detect a target molecule on a solid surface directly by spraying biosensing molecules. First, a filter paper as a model of a solid surface was spotted with a target nucleic acid. Then, the biosensing molecule, a DNA-nano-tweezers-structure that exhibits a peroxidase activity recognizing the target, was sprayed with hemin as a cofactor, followed by a second spray containing luminol as a substrate for a light-glowing signal. Finally, an image was recorded using a smartphone. The images revealed that only the places where the corresponding targets were spotted glowed a blue light.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116003"},"PeriodicalIF":2.5,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145407907","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advancing a sensitive method for measuring mitochondrial ATP production in small muscle biopsy samples","authors":"Rolf Wibom ,&nbsp;David Alsina ,&nbsp;Karin Naess ,&nbsp;Martin Engvall ,&nbsp;Christoph Freyer ,&nbsp;Anna Wedell ,&nbsp;Anna Wredenberg","doi":"10.1016/j.ab.2025.116004","DOIUrl":"10.1016/j.ab.2025.116004","url":null,"abstract":"<div><div>We present an optimised luminometric method for measuring muscle mitochondrial ATP production rate (MAPR), adapted to a 96-well microplate format. The enhanced assay enables quantification of ATP production from 12 or more substrate combinations within 15 min, using only 10 μL of isolated mitochondria. The method demonstrates high accuracy and precision, with a validated measurement range of 0.3–70 nmol/min/L. To support clinical interpretation, a reference dataset was established from 92 individuals aged seven months to 79 years. All these individuals were referred for muscle biopsy but were subsequently deemed unlikely to have a mitochondrial disorder following comprehensive clinical evaluation. An overview of the current version of our assays for oxidative phosphorylation (OXPHOS) enzymes is also provided.</div><div>As proof of concept, we present three patients carrying pathogenic variants in mitochondrial DNA (<em>ATP6</em> and <em>MT-TL1</em>) and the nuclear <em>PDHA1</em> gene. All exhibited decreased MAPR with one or more substrates, along with additional clinical, biochemical, and morphological features consistent with mitochondrial disease.</div><div>Furthermore, we illustrate the age-dependent development of MAPR in muscle across the human lifespan, demonstrating a 60–80 % higher maximal capacity for oxidative ATP production in adults compared with young children. In contrast, MAPR supported by fatty acid-derived substrates remains unchanged over the same period.</div><div>In conclusion, the improved MAPR assay offers a robust and efficient tool for assessing mitochondrial function in both clinical diagnostics and research. Its high-throughput format and reliable performance make it particularly well-suited for the investigation of suspected mitochondrial disorders.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116004"},"PeriodicalIF":2.5,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145420885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}