Analytical biochemistry最新文献_第4页

Differential binding of copper and zinc to a TDP-43 RNA recognition motif decapeptide and disulfide formation at residues C173/5 revealed by ESI-MS/MS ESI-MS/MS显示铜和锌与TDP-43 RNA识别基序十肽的差异结合和残基C173/5的二硫形成。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-12-13 DOI: 10.1016/j.ab.2025.116031

Josephine Esposto , Naomi L. Stock , Robert J. Huber , Sanela Martic

Copper (Cu) and zinc (Zn) metal ions play important roles in the proper functioning and localization of neurological proteins, such as transactive response DNA-binding protein 43 (TDP-43), which is linked to amyotrophic lateral sclerosis (ALS). Previous experimental and computational studies have identified putative Zn-binding regions within the RNA recognition motif 1 (RRM1) of TDP-43. However, Cu-binding interactions have been less explored despite their redox activity in regulating thiol (C173/175) conversion to disulfide within the RRM1 domain, influencing protein structure and function. Herein, the structural characterization and fragmentation pattern analysis of a TDP-43 decapeptide (166-HMIDGRWCDC-175), within RRM1, coordinated to Cu(II) and Zn(II) ions using electrospray ionization tandem mass spectrometry (ESI-MS/MS) was conducted under non-denaturing conditions. Higher-energy collision dissociation (HCD) fragmentation analysis identified that Cu(II) prefers His/Met residues, while Zn(II) was weakly coordinated to various binding sites in the peptide, specifically His, Met, Glu, Cys, Trp and Asp residues. Computational modeling using a metal ion binding server (MIB2) confirmed the binding sites and coordination sphere of metal-peptide complexes. No significant coordination to C173 and C175 was observed with Cu or Zn, as identified by using a double Cys mutant peptide. A complete thiol-to-disulfide conversion was observed in the presence of Cu(II)/(I) only, which was confirmed by the comparison of a preformed intramolecular disulfide peptide. Overall, unique differential coordination environments were observed for each metal ion with the peptide. The study provides new insights into metal ion interactions with TDP-43 RRM1 peptide, leading to a greater understanding of metal homeostasis in TDP-43 protein biochemistry and neurodegeneration.

铜（Cu）和锌（Zn）金属离子在神经系统蛋白的正常功能和定位中发挥重要作用，例如与肌萎缩性侧索硬化症（ALS）有关的交互反应dna结合蛋白43 （TDP-43）。先前的实验和计算研究已经在TDP-43的RNA识别基序1 （RRM1）中确定了假定的锌结合区域。然而，尽管cu结合相互作用在调节硫醇（C173/175）在RRM1结构域中转化为二硫化合物，影响蛋白质结构和功能方面具有氧化还原活性，但对它们的探索较少。在非变性条件下，利用电喷雾串联质谱（ESI-MS/MS）对RRM1中与Cu（II）和Zn（II）离子配位的TDP-43十肽（166-HMIDGRWCDC-175）进行了结构表征和碎片化模式分析。高能碰撞解离（HCD）碎片化分析发现，Cu（II）倾向于His/Met残基，而Zn（II）与肽中的各种结合位点弱协同，特别是His、Met、Glu、Cys、Trp和Asp残基。利用金属离子结合服务器（MIB2）的计算模型确定了金属肽配合物的结合位点和配位球。通过双Cys突变肽鉴定，C173和C175与Cu或Zn没有明显的配位关系。仅在Cu(II)/(I)存在的情况下，观察到硫醇到二硫的完全转化，这一点通过对预形成的分子内二硫肽的比较得到证实。总的来说，观察到每个金属离子与肽的独特的差异配位环境。该研究为金属离子与TDP-43 RRM1肽的相互作用提供了新的见解，从而更好地了解TDP-43蛋白生物化学和神经退行性变中的金属稳态。

{"title":"Differential binding of copper and zinc to a TDP-43 RNA recognition motif decapeptide and disulfide formation at residues C173/5 revealed by ESI-MS/MS","authors":"Josephine Esposto , Naomi L. Stock , Robert J. Huber , Sanela Martic","doi":"10.1016/j.ab.2025.116031","DOIUrl":"10.1016/j.ab.2025.116031","url":null,"abstract":"<div><div>Copper (Cu) and zinc (Zn) metal ions play important roles in the proper functioning and localization of neurological proteins, such as transactive response DNA-binding protein 43 (TDP-43), which is linked to amyotrophic lateral sclerosis (ALS). Previous experimental and computational studies have identified putative Zn-binding regions within the RNA recognition motif 1 (RRM1) of TDP-43. However, Cu-binding interactions have been less explored despite their redox activity in regulating thiol (C173/175) conversion to disulfide within the RRM1 domain, influencing protein structure and function. Herein, the structural characterization and fragmentation pattern analysis of a TDP-43 decapeptide (166-HMIDGRWCDC-175), within RRM1, coordinated to Cu(II) and Zn(II) ions using electrospray ionization tandem mass spectrometry (ESI-MS/MS) was conducted under non-denaturing conditions. Higher-energy collision dissociation (HCD) fragmentation analysis identified that Cu(II) prefers His/Met residues, while Zn(II) was weakly coordinated to various binding sites in the peptide, specifically His, Met, Glu, Cys, Trp and Asp residues. Computational modeling using a metal ion binding server (MIB2) confirmed the binding sites and coordination sphere of metal-peptide complexes. No significant coordination to C173 and C175 was observed with Cu or Zn, as identified by using a double Cys mutant peptide. A complete thiol-to-disulfide conversion was observed in the presence of Cu(II)/(I) only, which was confirmed by the comparison of a preformed intramolecular disulfide peptide. Overall, unique differential coordination environments were observed for each metal ion with the peptide. The study provides new insights into metal ion interactions with TDP-43 RRM1 peptide, leading to a greater understanding of metal homeostasis in TDP-43 protein biochemistry and neurodegeneration.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"710 ","pages":"Article 116031"},"PeriodicalIF":2.5,"publicationDate":"2025-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145762025","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}

引用次数: 0

Identification of the function of the metabolite repair enzyme Nit1: the story of a collaboration with Arthur Cooper 鉴定代谢物修复酶Nit1的功能：与Arthur Cooper合作的故事。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-12-11 DOI: 10.1016/j.ab.2025.116032

Emile Van Schaftingen , Alessio Peracchi , Maria Veiga-da-Cunha

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.

Nit1和Nit2最初是在癌症研究的背景下被发现的，是由推定的（抗）癌基因编码的蛋白质。然而，细菌中同源蛋白的存在表明它们可能是具有基本代谢功能的酶。我们的团队，在与Arthur Cooper和他的合作者相互作用的同时，为揭示这些作用做出了贡献：Nit2在2009年被确定为ω-氨基酶，这种酶水解α-酮戊二酸和α-酮琥珀酸的ω酰胺，在一些不可逆的转氨化反应中产生谷氨酰胺和天冬酰胺的“脱胺”衍生物。随后，在2017年，我们发现Nit1作为代谢物修复酶起作用。具体来说，Nit1有效地水解脱氨谷胱甘肽（dGSH），这是一种由各种经典转氨酶的副作用产生的无功能副产物。这种修复功能可以防止无用的代谢物dGSH的积累。最近的发现强调了Nit1的生理意义，将其在人类中的缺乏与神经系统疾病联系起来。

{"title":"Identification of the function of the metabolite repair enzyme Nit1: the story of a collaboration with Arthur Cooper","authors":"Emile Van Schaftingen , Alessio Peracchi , 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":"2025-12-11","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}

引用次数: 0

Multi-disciplinary combination to explore the laxation mechanism of Rhei Radix et Rhizoma and Magnoliae Officinalis Cortex 多学科结合探讨大黄、厚朴通便作用机制

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-12-09 DOI: 10.1016/j.ab.2025.116030

Meiyu Wan, Yu Wang, Meijuan Liu, Wenwen Zhou, Xiaoxiao Zhang, Mingyang Wang, Shu Jiang, Erxin Shang, Jinao Duan

Background

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.

Materials and methods

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.

Results

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.

Conclusion

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.

大黄（RR）与厚朴皮质（MO）联合治疗便秘已有数千年的历史。然而，两种草药协同作用通便作用的机制尚不清楚。材料与方法本研究成功建立盐酸洛哌丁胺便秘大鼠模型，系统、全面评价RR和MO对便秘的改善作用。结合网络药理学、分子对接和基于UPLC-Q-TOF/ ms的代谢组学，揭示RR和MO的潜在配合性机制。MetaboAnalyst和MetaScape联合分析网络药理学靶点和代谢组学代谢物所涉及的代谢途径，选择关键代谢途径。结果在生理生化水平上，RR和MO的通便效果明显优于单一中药。网络药理学和分子对接结果显示，RR和MO可通过多组分、多靶点、多途径改善便秘。代谢组学研究表明，RR和MO联合治疗可明显逆转模型大鼠血浆中紊乱的代谢谱。此外，综合分析9种代谢物和124个靶点后，我们选择花生四烯酸作为最关键的途径。结果表明，该中药对可通过花生四烯酸途径缓解便秘，并抑制该途径中PTGS2和PLA2G2A蛋白水平。结论本研究从生物信息学和代谢组学角度揭示了RR与MO联合用药对便秘的协同改善作用。此外，多组学的整合方法有助于探索中药的潜在配伍机制。

{"title":"Multi-disciplinary combination to explore the laxation mechanism of Rhei Radix et Rhizoma and Magnoliae Officinalis Cortex","authors":"Meiyu Wan, Yu Wang, Meijuan Liu, Wenwen Zhou, Xiaoxiao Zhang, Mingyang Wang, Shu Jiang, Erxin Shang, 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":"2025-12-09","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}

引用次数: 0

Classification prediction of drug target binding affinity based on the MolrProtTrans model 基于MolrProtTrans模型的药物靶点结合亲和力分类预测。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-12-04 DOI: 10.1016/j.ab.2025.116029

Yicun Lin, Yuanfeng Li, Wei Sun, Jian Wang

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.

预测药物-靶标相互作用对虚拟药物筛选至关重要。虽然许多模型预测了小分子与蛋白质之间的结合亲和力，但它们往往过分强调分子特征，而忽略了重要的蛋白质特征，导致预测有偏差。传统的深度学习模型，如transformerCPI，在G蛋白偶联受体（GPCR）数据集的标签反演等任务中表现有限。为了解决这个问题，本研究提出了一种基于转换器的增强模型，该模型集成了分子和蛋白质信息。通过利用Molr和ProtTrans网络进行特征提取，并将转置注意机制与三损失自监督学习方法相结合，该模型提高了预测精度。实验结果表明，该模型在GPCR标记反演数据集上实现了0.81的曲线下面积（Area Under the Curve， AUC），在人类目标数据集上实现了0.92的曲线下面积（Area Under the Curve， AUC），在数值上高于TransformerCPI和几种基线方法。这些观察结果表明，在我们的实验环境中，性能有所提高，为推进虚拟药物筛选和药物发现提供了广阔的前景。

{"title":"Classification prediction of drug target binding affinity based on the MolrProtTrans model","authors":"Yicun Lin, Yuanfeng Li, Wei Sun, 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":"2025-12-04","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}

引用次数: 0

Single-Entity Dual-Emissive MOF Platform for Reliable Ratiometric Point-of-Care Detection of Amoxicillin Residues 单实体双发射MOF平台可靠的比率点检测阿莫西林残留

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-12-03 DOI: 10.1016/j.ab.2025.116028

Sameera Sh. Mohammed Ameen , Fiasal K. Algethami , Khalid M. Omer , Idrees B. Qader , Hemn A. Qader

In fluorescence-based sensing, self-referencing ratiometric analysis offers a significant advantage over external referencing by integrating both the probe and reference signals within a single material, rather than relying on two separate components. This intrinsic approach eliminates the need for additional reference dyes or materials, which can introduce inconsistencies due to variations in concentration, uneven dispersion, or environmental instability. Self-referencing materials provide a built-in correction mechanism, enhancing detection accuracy, reliability, and reproducibility while minimizing background interference. Despite their advantages, the design and synthesis of dual-emitting metal-organic frameworks (MOFs) with self-referencing capabilities remain rare and challenging. In this study, we introduce a novel Eu-based MOF with intrinsic dual-state, dual-emission properties, exhibiting distinct blue and red fluorescence in both liquid and solid states. The blue emission arises from the coordination-induced emission of the free, non-emissive ligand within the MOF structure. Interestingly, the red emission is selectively quenched by amoxicillin (AMX), while the blue fluorescence remains unaffected. This unique dual-emission feature enables a ratiometric sensing platform without requiring an external reference, ensuring greater stability, accuracy, and ease of use. With a linear detection range of 8.0–218 μM and a limit of detection of 0.354 μM, this Eu-MOF offers a robust and selective AMX sensing strategy. Additionally, a smartphone-assisted visual detection method using RGB analysis via the Color Grab App was developed, enabling portable and on-site quantification. This self-referencing Eu-MOF is inherently stable, recyclable, providing consistent signals and making it highly effective for pharmaceutical applications.

在基于荧光的传感中，自参考比率分析通过在单个材料中集成探针和参考信号而不是依赖于两个单独的组件，比外部参考具有显著的优势。这种固有的方法消除了额外的参考染料或材料的需要，这些参考染料或材料可能由于浓度变化、分散不均匀或环境不稳定而导致不一致。自参考材料提供内置校正机制，提高检测精度，可靠性和再现性，同时最大限度地减少背景干扰。尽管具有这些优点，但设计和合成具有自参考能力的双发射金属有机框架（mof）仍然很少见和具有挑战性。在这项研究中，我们引入了一种新型的铕基MOF，它具有固有的双态、双发射特性，在液体和固体状态下都表现出明显的蓝色和红色荧光。蓝色发射是由MOF结构中自由的非发射配体的配位引起的。有趣的是，红色荧光被阿莫西林（AMX）选择性猝灭，而蓝色荧光不受影响。这种独特的双发射功能使比率传感平台无需外部参考，确保更高的稳定性，准确性和易用性。该Eu-MOF的线性检测范围为8.0-218 μM，检测限为0.354 μM，提供了鲁棒性和选择性的AMX检测策略。此外，开发了一种智能手机辅助的视觉检测方法，通过Color Grab App使用RGB分析，实现便携式和现场定量。这种自我参考的Eu-MOF本质上是稳定的，可回收的，提供一致的信号，使其在制药应用中非常有效。

{"title":"Single-Entity Dual-Emissive MOF Platform for Reliable Ratiometric Point-of-Care Detection of Amoxicillin Residues","authors":"Sameera Sh. Mohammed Ameen , Fiasal K. Algethami , Khalid M. Omer , Idrees B. Qader , Hemn A. Qader","doi":"10.1016/j.ab.2025.116028","DOIUrl":"10.1016/j.ab.2025.116028","url":null,"abstract":"<div><div>In fluorescence-based sensing, self-referencing ratiometric analysis offers a significant advantage over external referencing by integrating both the probe and reference signals within a single material, rather than relying on two separate components. This intrinsic approach eliminates the need for additional reference dyes or materials, which can introduce inconsistencies due to variations in concentration, uneven dispersion, or environmental instability. Self-referencing materials provide a built-in correction mechanism, enhancing detection accuracy, reliability, and reproducibility while minimizing background interference. Despite their advantages, the design and synthesis of dual-emitting metal-organic frameworks (MOFs) with self-referencing capabilities remain rare and challenging. In this study, we introduce a novel Eu-based MOF with intrinsic dual-state, dual-emission properties, exhibiting distinct blue and red fluorescence in both liquid and solid states. The blue emission arises from the coordination-induced emission of the free, non-emissive ligand within the MOF structure. Interestingly, the red emission is selectively quenched by amoxicillin (AMX), while the blue fluorescence remains unaffected. This unique dual-emission feature enables a ratiometric sensing platform without requiring an external reference, ensuring greater stability, accuracy, and ease of use. With a linear detection range of 8.0–218 μM and a limit of detection of 0.354 μM, this Eu-MOF offers a robust and selective AMX sensing strategy. Additionally, a smartphone-assisted visual detection method using RGB analysis via the Color Grab App was developed, enabling portable and on-site quantification. This self-referencing Eu-MOF is inherently stable, recyclable, providing consistent signals and making it highly effective for pharmaceutical applications.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"710 ","pages":"Article 116028"},"PeriodicalIF":2.5,"publicationDate":"2025-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145684009","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}

引用次数: 0

Optimized purification workflow for advanced eEF1A studies: Impact of His-tagging on stability, functionality, and yield 先进eEF1A研究的优化纯化工作流程：his标记对稳定性，功能和产量的影响。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-12-02 DOI: 10.1016/j.ab.2025.116017

Pernille Nedergaard Madsen, Pernille Baden Jørgensen, Mille Varisbøl Clausen, Charlotte Rohde Knudsen

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 Saccharomyces cerevisiae 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 in vitro, 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.

真核延伸因子1A （eEF1A）在翻译中起着至关重要的作用，它将氨基酰基trna传递到核糖体A位点。虽然它的细菌对应物，伸长因子热不稳定（EF-Tu），已经被广泛研究，但eEF1A的规范和非规范作用仍然知之甚少，部分原因是缺乏有效的纯化方法。本研究优化了一种基于亲和层析的酿酒酵母eEF1A方案，评估了N端和c端his标记对稳定性、功能、纯化和产量的影响。在体外实验中，四个残基的n端缺失会破坏三元复合物的形成，而在同一位置的his标记则不会。然而，纳米差示扫描荧光法（NanoDSF）显示，n端his -标签破坏了eEF1A的稳定性，而c端his -标签保持了其完整性并提高了收率。此外，将甘油浓度从25%降低到10%加速了纯化，而不影响稳定性或tRNA结合。这种优化的c端his标签协议为研究eEF1A的功能和动态特性提供了一种简化的方法。

{"title":"Optimized purification workflow for advanced eEF1A studies: Impact of His-tagging on stability, functionality, and yield","authors":"Pernille Nedergaard Madsen, Pernille Baden Jørgensen, Mille Varisbøl Clausen, 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":"2025-12-02","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}

引用次数: 0

Advances in the detection of azodicarbonamide and the metabolic product semicarbazide 偶氮二甲酰胺及其代谢产物氨基脲的检测研究进展。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-11-27 DOI: 10.1016/j.ab.2025.116018

Zijuan Miao , Bowen Kan , Pan Liu , Yongming Guo

Azodicarbonamide (ADA) is widely used as a foaming and oxidizing agent in the food and plastic industries. However, long-term exposure to it could cause serious health problems. Its metabolic product, semicarbazide (SEM), has raised health concerns due to findings of potential carcinogenicity and genotoxicity in some studies, though the evidence is context-dependent and sometimes conflicting. Therefore, the development of efficient, sensitive, and rapid detection methods for ADA and SEM is of great significance for food safety assurance. This review systematically summarizes recent advances in detection techniques for ADA and SEM, including high-performance liquid chromatography, capillary electrophoresis, Raman spectroscopy, immunoassays, electrochemical methods, near-infrared spectroscopy, colorimetry, fluorescence, chemiluminescence, photoacoustic, and photothermal detection. Subsequently, the sensitivity, selectivity, applicability, and limitations of these methods are compared and analyzed. Lastly, the challenges and future research trends are discussed. This review will pave the way for the development of advanced sensing strategies for ADA and SEM.

偶氮二甲酰胺（ADA）在食品和塑料工业中广泛用作泡沫剂和氧化剂。然而，长期接触它可能会导致严重的健康问题。由于在一些研究中发现了潜在的致癌性和遗传毒性，其代谢产物氨基脲（SEM）引起了健康问题，尽管证据依赖于环境，有时相互矛盾。因此，开发高效、灵敏、快速的ADA和SEM检测方法对保证食品安全具有重要意义。本文系统地综述了ADA和SEM检测技术的最新进展，包括高效液相色谱、毛细管电泳、拉曼光谱、免疫分析、电化学方法、近红外光谱、比色法、荧光、化学发光、光声和光热检测。随后，对这些方法的灵敏度、选择性、适用性和局限性进行了比较分析。最后，对未来的研究趋势和面临的挑战进行了讨论。本综述将为ADA和SEM的先进传感策略的发展铺平道路。

{"title":"Advances in the detection of azodicarbonamide and the metabolic product semicarbazide","authors":"Zijuan Miao , Bowen Kan , Pan Liu , Yongming Guo","doi":"10.1016/j.ab.2025.116018","DOIUrl":"10.1016/j.ab.2025.116018","url":null,"abstract":"<div><div>Azodicarbonamide (ADA) is widely used as a foaming and oxidizing agent in the food and plastic industries. However, long-term exposure to it could cause serious health problems. Its metabolic product, semicarbazide (SEM), has raised health concerns due to findings of potential carcinogenicity and genotoxicity in some studies, though the evidence is context-dependent and sometimes conflicting. Therefore, the development of efficient, sensitive, and rapid detection methods for ADA and SEM is of great significance for food safety assurance. This review systematically summarizes recent advances in detection techniques for ADA and SEM, including high-performance liquid chromatography, capillary electrophoresis, Raman spectroscopy, immunoassays, electrochemical methods, near-infrared spectroscopy, colorimetry, fluorescence, chemiluminescence, photoacoustic, and photothermal detection. Subsequently, the sensitivity, selectivity, applicability, and limitations of these methods are compared and analyzed. Lastly, the challenges and future research trends are discussed. This review will pave the way for the development of advanced sensing strategies for ADA and SEM.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116018"},"PeriodicalIF":2.5,"publicationDate":"2025-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145627607","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}

引用次数: 0

Structural, optical, and electrochemical characterization of TiO2-x thin films for non-enzymatic amperometric glucose sensing 用于非酶促电流葡萄糖传感的TiO2-x薄膜的结构、光学和电化学表征。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-11-22 DOI: 10.1016/j.ab.2025.116016

J.K. Olarte Villamizar, M. Zapata Torres, N. Cruz González, G. Silva Galindo

This work presents a novel approach to non-enzymatic glucose sensing based on oxygen-deficient titanium dioxide (TiO_2-x) thin films deposited on metallic titanium substrates. The TiO_2-x films were fabricated via RF magnetron reactive sputtering using a titanium target, and their structural, morphological, optical, and electrochemical properties were comprehensively characterized through X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), UV–Vis spectroscopy, and electrochemical techniques. The electrochemical performance was evaluated using cyclic voltammetry (CV) and chronoamperometry. The CV results revealed that the oxidation in phosphate buffer solution occurs at an applied potential of −0.54 V, increasing with a glucose concentration. Chronoamperometric analysis demonstrated a high sensitivity of 27.55 μA cm⁻² mM⁻¹ and a low detection limit of 0.11 mM. The sensor also exhibited a linear dynamic range from 0.2 mM to 2.4 mM and excellent selectivity against common interfering species, including histidine (His), cysteine (Cys), uric acid (UA), ascorbic acid (AA), and sucrose. These findings highlight the potential of Ti/TiO_2-x electrodes as efficient, stable, and selective platforms for developing non-enzymatic glucose sensors suitable for biomedical and wearable applications, with possible use in detecting glucose concentrations in sweat (0.27 mM–1 mM) and saliva (0.23 mM–1.78 mM).

这项工作提出了一种基于沉积在金属钛衬底上的缺氧二氧化钛（TiO2-x）薄膜的非酶促葡萄糖传感新方法。以钛为靶材，采用射频磁控反应溅射法制备TiO2-x薄膜，并通过x射线衍射（XRD）、扫描电镜（SEM）、x射线光电子能谱（XPS）、紫外可见光谱（UV-Vis）和电化学技术对其结构、形貌、光学和电化学性能进行了全面表征。采用循环伏安法和计时安培法对其电化学性能进行了评价。CV结果表明，磷酸缓冲溶液中的氧化发生在-0.54 V的电位下，随着葡萄糖浓度的增加而增加。该传感器的灵敏度为27.55 μA cm-2·mM-1，检出限为0.11 mM，线性动态范围为0.2 ~ 2.4 mM，对组氨酸（His）、半胱氨酸（Cys）、尿酸（UA）、抗坏血酸（AA）和糖等常见干扰物质具有良好的选择性。这些发现突出了Ti/TiO2-x电极作为高效、稳定和选择性平台的潜力，可用于开发适合生物医学和可穿戴应用的非酶葡萄糖传感器，可能用于检测汗液（0.27 mM-1 mM）和唾液（0.23 mM-1.78 mM）中的葡萄糖浓度。

{"title":"Structural, optical, and electrochemical characterization of TiO2-x thin films for non-enzymatic amperometric glucose sensing","authors":"J.K. Olarte Villamizar, M. Zapata Torres, N. Cruz González, G. Silva Galindo","doi":"10.1016/j.ab.2025.116016","DOIUrl":"10.1016/j.ab.2025.116016","url":null,"abstract":"<div><div>This work presents a novel approach to non-enzymatic glucose sensing based on oxygen-deficient titanium dioxide (TiO<sub>2-x</sub>) thin films deposited on metallic titanium substrates. The TiO<sub>2-x</sub> films were fabricated via RF magnetron reactive sputtering using a titanium target, and their structural, morphological, optical, and electrochemical properties were comprehensively characterized through X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), UV–Vis spectroscopy, and electrochemical techniques. The electrochemical performance was evaluated using cyclic voltammetry (CV) and chronoamperometry. The CV results revealed that the oxidation in phosphate buffer solution occurs at an applied potential of −0.54 V, increasing with a glucose concentration. Chronoamperometric analysis demonstrated a high sensitivity of 27.55 μA cm<sup>−2</sup> mM<sup>−1</sup> and a low detection limit of 0.11 mM. The sensor also exhibited a linear dynamic range from 0.2 mM to 2.4 mM and excellent selectivity against common interfering species, including histidine (His), cysteine (Cys), uric acid (UA), ascorbic acid (AA), and sucrose. These findings highlight the potential of Ti/TiO<sub>2-x</sub> electrodes as efficient, stable, and selective platforms for developing non-enzymatic glucose sensors suitable for biomedical and wearable applications, with possible use in detecting glucose concentrations in sweat (0.27 mM–1 mM) and saliva (0.23 mM–1.78 mM).</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116016"},"PeriodicalIF":2.5,"publicationDate":"2025-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145595681","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}

引用次数: 0

Affinity-based selection of anti-Feline Leukaemia Virus p27 monoclonal antibodies for efficient lateral flow assay development 基于亲和选择的抗猫白血病病毒p27单克隆抗体用于高效的横向流动试验开发。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-11-19 DOI: 10.1016/j.ab.2025.116013

Hadyn Duncan , Elisabeth Domingo-Contreras , Sotirios Athanasiou , Rosario Fernandez-Godino , Francisco Castillo , Ana Camacho

Traditional diagnostic methods like RT-PCR and proviral DNA PCR have limitations, underscoring the need for swift, sensitive alternatives such as lateral flow immunoassays (LFIA). Yet, efficiently identifying monoclonal antibody (mAb) pairs for robust ternary antigen-antibody interactions remains a universal bottleneck, as conventional ELISA screening is costly and may not accurately reflect native binding. Here, we combined ELISA with Spectral Shift Technology (SST) to minimise reagent use and comprehensively profile anti-FeLV p27 mAbs. This orthogonal approach enabled rapid selection of two low nanomolar mAbs with different epitope targets that form a ternary complex, which translates into a sensitive detection of the FeLV p27 in a LFIA prototype. Our screening methodology supports resource-efficient LFIA development for FeLV and extends to other significant animal and human pathogens like Monkeypox or Helicobacter pylori, improving diagnostics and quality control.

传统的诊断方法，如RT-PCR和前病毒DNA PCR有局限性，强调需要快速、敏感的替代方法，如侧流免疫测定（LFIA）。然而，有效地识别单克隆抗体（mAb）对的强效三元抗原-抗体相互作用仍然是一个普遍的瓶颈，因为传统的ELISA筛选成本高昂，可能无法准确反映天然结合。在这里，我们将ELISA与光谱移位技术（SST）相结合，以减少试剂的使用，并全面分析抗felv p27单克隆抗体。这种正交方法能够快速选择具有不同表位靶点的两种低纳摩尔单抗，形成三元复合物，从而在LFIA原型中转化为对FeLV p27的敏感检测。我们的筛选方法支持对FeLV进行资源高效的LFIA开发，并扩展到其他重要的动物和人类病原体，如猴痘或幽门螺杆菌，从而提高诊断和质量控制。

{"title":"Affinity-based selection of anti-Feline Leukaemia Virus p27 monoclonal antibodies for efficient lateral flow assay development","authors":"Hadyn Duncan , Elisabeth Domingo-Contreras , Sotirios Athanasiou , Rosario Fernandez-Godino , Francisco Castillo , Ana Camacho","doi":"10.1016/j.ab.2025.116013","DOIUrl":"10.1016/j.ab.2025.116013","url":null,"abstract":"<div><div>Traditional diagnostic methods like RT-PCR and proviral DNA PCR have limitations, underscoring the need for swift, sensitive alternatives such as lateral flow immunoassays (LFIA). Yet, efficiently identifying monoclonal antibody (mAb) pairs for robust ternary antigen-antibody interactions remains a universal bottleneck, as conventional ELISA screening is costly and may not accurately reflect native binding. Here, we combined ELISA with Spectral Shift Technology (SST) to minimise reagent use and comprehensively profile anti-FeLV p27 mAbs. This orthogonal approach enabled rapid selection of two low nanomolar mAbs with different epitope targets that form a ternary complex, which translates into a sensitive detection of the FeLV p27 in a LFIA prototype. Our screening methodology supports resource-efficient LFIA development for FeLV and extends to other significant animal and human pathogens like Monkeypox or <em>Helicobacter pylori</em>, improving diagnostics and quality control.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116013"},"PeriodicalIF":2.5,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145562491","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}

引用次数: 0

One-step purification of a bioactive PAK1-derived peptide 生物活性pak1衍生肽的一步纯化

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-11-19 DOI: 10.1016/j.ab.2025.116014

Djamali Muhoza , Emily P. Esquivel , Stacy R. Hunter , Patience S. Okoto , Thallapuranam K.S. Kumar , Paul D. Adams

The serine/threonine kinase PAK1 serves as a mediator of cytoskeletal reorganization and cancer-related signaling downstream of the small GTPases. Due to the challenges in purifying PAK1 complexes, a 46-residue peptide from PAK1, is widely used to study PAK1-Cdc42 signaling. Traditionally, this purification involved multi-step chromatography of recombinant GST-PBD46 complexes, yielding approximately 1 mg per 1.5 L culture. In this study, a 30 min heat treatment step after thrombin cleavage was used to precipitate GST while leaving pure PBD46 in solution. This step eliminated the need for further affinity and size-exclusion chromatography steps. This improved protocol produces proteins with a 6.5-fold higher yield, halves the purification processing time, and produces peptides with ≥95 % purity. Mass spectrometry, CD, fluorescence, and ¹H–¹⁵N HSQC NMR confirmed the heat-purified PBD46's identity, structure, folding, and binding to Cdc42. The method also successfully separated other small peptides (e.g., ACK1) but not larger folded proteins. This rapid and scalable approach facilitates peptide production and biochemical studies without compromising the structural or functional integrity. We believe that the method described herein is applicable to other stable GST-fused recombinant proteins.

丝氨酸/苏氨酸激酶PAK1是小gtpase下游细胞骨架重组和癌症相关信号传导的中介。由于PAK1复合物的纯化困难，PAK1的46位残基肽被广泛用于研究PAK1- cdc42信号转导。传统上，这种纯化涉及重组GST-PBD46复合物的多步色谱，每1.5 L培养产生约1 mg。在本研究中，凝血酶裂解后30分钟的热处理步骤沉淀GST，同时将纯PBD46留在溶液中。这一步骤消除了进一步亲和层析和尺寸排除层析步骤的需要。该改进方案生产的蛋白质产量提高6.5倍，纯化处理时间缩短一半，并生产纯度≥95%的肽。质谱、CD、荧光和1H-15N HSQC NMR证实了热纯化PBD46的身份、结构、折叠和与Cdc42的结合。该方法还成功地分离了其他小肽（如ACK1），但不能分离较大的折叠蛋白。这种快速和可扩展的方法促进了肽的生产和生化研究，而不影响结构或功能的完整性。我们相信本文描述的方法适用于其他稳定的gst融合重组蛋白。

{"title":"One-step purification of a bioactive PAK1-derived peptide","authors":"Djamali Muhoza , Emily P. Esquivel , Stacy R. Hunter , Patience S. Okoto , Thallapuranam K.S. Kumar , Paul D. Adams","doi":"10.1016/j.ab.2025.116014","DOIUrl":"10.1016/j.ab.2025.116014","url":null,"abstract":"<div><div>The serine/threonine kinase PAK1 serves as a mediator of cytoskeletal reorganization and cancer-related signaling downstream of the small GTPases. Due to the challenges in purifying PAK1 complexes, a 46-residue peptide from PAK1, is widely used to study PAK1-Cdc42 signaling. Traditionally, this purification involved multi-step chromatography of recombinant GST-PBD46 complexes, yielding approximately 1 mg per 1.5 L culture. In this study, a 30 min heat treatment step after thrombin cleavage was used to precipitate GST while leaving pure PBD46 in solution. This step eliminated the need for further affinity and size-exclusion chromatography steps. This improved protocol produces proteins with a 6.5-fold higher yield, halves the purification processing time, and produces peptides with ≥95 % purity. Mass spectrometry, CD, fluorescence, and <sup>1</sup>H–<sup>15</sup>N HSQC NMR confirmed the heat-purified PBD46's identity, structure, folding, and binding to Cdc42. The method also successfully separated other small peptides (e.g., ACK1) but not larger folded proteins. This rapid and scalable approach facilitates peptide production and biochemical studies without compromising the structural or functional integrity. We believe that the method described herein is applicable to other stable GST-fused recombinant proteins.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116014"},"PeriodicalIF":2.5,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145562406","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}

引用次数: 0