ELECTROPHORESIS最新文献

{"title":"Enabling icIEF Peak Identification of AAV Capsid Proteins by Fractionation on MauriceFlex and Subsequent Analysis by LC–MS","authors":"Will McElroy,&nbsp;Sisi Huang,&nbsp;Xiaoping He,&nbsp;Cheng Zhou,&nbsp;Christopher D. Heger,&nbsp;Thomas W. Powers,&nbsp;Melissa M. Anderson,&nbsp;Courtney Sloan,&nbsp;Thomas F. Lerch","doi":"10.1002/elps.202400201","DOIUrl":"10.1002/elps.202400201","url":null,"abstract":"<p>A significant limitation of imaged capillary electric focusing (icIEF) is the inability to identify and characterize specific species in the electropherogram. This has led to the development of complementary ion-exchange chromatography (IEX)-based methods that are amenable to either fraction collection and subsequent characterization or online IEX coupled to mass spectrometry. To overcome this limitation while maintaining the use of icIEF, novel approaches, including an icIEF separation and fractionation technology (MauriceFlex, ProteinSimple), have been developed. This approach enables the fractionation of various icIEF peaks, which can then be characterized by mass spectrometry to confirm the identity of the separated charged species. Herein, the MauriceFlex technology was applied to adeno-associated viral (AAV) gene therapy products, which contain a DNA transgene packaged into a protein capsid and have shown tremendous therapeutic potential in recent years. Utilizing the MauriceFlex system, we developed an approach for the separation of charged species from AAV capsid viral proteins (VP) by icIEF and subsequent characterization by liquid chromatography and mass spectrometry (LC–MS). When applying the same sample preparation, charge profiles of AAV capsid proteins on the MauriceFlex instrument were demonstrated to be consistent with those from the original Maurice platform, the industrial gold standard. Optimization of the VP icIEF fractionation method required the development of a method for low concentration samples, optimization of mobilization conditions, enhancement of fraction recovery, and maintenance of protein stability post fractionation. Herein, we were able to successfully collect charge-separated VP fraction samples and subsequently analyze them by MS analysis. In addition, a workflow for AAV capsid protein characterization based on icIEF separation and fractionation coupled with downstream LC–MS has been established for the confirmation of VP identity and additional characterization of capsid protein heterogeneity.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"46 1-2","pages":"22-33"},"PeriodicalIF":3.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11773307/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143001742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spatio-Temporal Change of Skin and Oral Microbiota: A Longitudinal Study of Microbial Diversity and Stability","authors":"Han Zhang,&nbsp;Anqi Chen,&nbsp;Shilin Li,&nbsp;Kaiqin Chen,&nbsp;Xuechun You,&nbsp;Yingnan Bian,&nbsp;Chengtao Li,&nbsp;Shiquan Liu,&nbsp;Jiang Huang,&nbsp;Suhua Zhang","doi":"10.1002/elps.202400160","DOIUrl":"10.1002/elps.202400160","url":null,"abstract":"<div>\u0000 \u0000 <p>The human skin and oral cavity harbor complex microbial communities, which exist in dynamic equilibrium with the host's physiological state and the external environment. This study investigates the microbial atlas of human skin and oral cavities using samples collected over a 10-month period, aiming to assess how both internal and external factors influence the human microbiome. We examined bacterial community diversity and stability across various body sites, including palm and nasal skin, saliva, and oral epithelial cells, during environmental changes and a COVID-19 pandemic. The skin microbiome was confirmed to display spatial and temporal stability compared to the oral microbiome, particularly the oral epithelium, which was susceptible to changes in the host's physiological state and immune response. Moreover, significant differences in the microbial community structure among the 4 sample types were observed, and 87 distinct bacteria biomarkers were identified. The random forest prediction model achieved an overall prediction accuracy of 95.24% across the four types of samples studied. Additionally, nasal skin samples showed significant promise for individual identification through profiling the skin microbiota. These findings highlight the potential of skin and oral microbiota as forensic markers for inferring body sites and identifying individuals. In summary, despite facing limitations such as a small cohort size and the need for broader validation, this research provides an overall perspective and initial insights for refining experimental designs and conducting in-depth research in various microbial research fields.</p>\u0000 </div>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"46 1-2","pages":"92-103"},"PeriodicalIF":3.0,"publicationDate":"2025-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142970151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Computer Simulation of Sulfated Cyclodextrin-Based Enantioselective Separation of Weak Bases With Partial, High-Concentration Filling of the Chiral Selector and Analyte Detection on the Cathodic Side.","authors":"Friederike A Sandbaumhüter, Wolfgang Thormann","doi":"10.1002/elps.202400213","DOIUrl":"https://doi.org/10.1002/elps.202400213","url":null,"abstract":"<p><p>Computer simulation was utilized to characterize the electrophoretic processes occurring during the enantioselective capillary electrophoresis-mass spectrometry (CE-MS) analysis of ketamine, norketamine, and hydroxynorketamine in a system with partial filling of the capillary with 19 mM (equals 5%) of highly sulfated γ-cyclodextrin (HS-γ-CD) and analyte detection on the cathodic side. Provided that the sample is applied without or with a small amount of the chiral selector, analytes become quickly focused and separated in the thereby formed HS-γ-CD gradient at the cathodic end of the sample compartment. This gradient broadens with time, remains stationary, and gradually reduces its span from the lower side due to diffusion such that analytes with high affinity to the anionic selector become released onto the other side of the focusing gradient where anionic migration and defocusing occur concomitantly. The analytes that remain focused until the migrating HS-γ-CD concentration boundary arrives at the cathodic end of the sample compartment become gradually released into the cathodic part and migrate in the absence of HS-γ-CD toward the detector. This behavior is dependent on the length of the HS-γ-CD zone in the cathodic part of the electrophoretic column, the initial sample zone length, and the sample matrix. The data presented reveal the possibility that only one of the enantiomers of an analyte migrates toward the detector, whereas the other is lost for the analysis, or that both enantiomers migrate toward the cathode but do not separate. Enantiomer separation followed by migration toward the cathode can only be achieved for analytes with rather low complexation constants, such as hydroxynorketamine assessed in this work, and is dependent on the slope of the HS-γ-CD focusing gradient. The gained insights illustrate that dynamic simulation is an indispensable tool to investigate electrophoretic processes of complex systems.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142970189","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative Proteomics of Resistant and Susceptible Strains of Frankliniella occidentalis to Abamectin","authors":"Zahra Gholami,&nbsp;Foad Fatehi,&nbsp;Fatemeh Habibpour Mehraban,&nbsp;Paul A. Haynes,&nbsp;Khalil Talebi Jahromi,&nbsp;Vahid Hosseininaveh,&nbsp;Hadi Mosallanejad,&nbsp;Pär K. Ingvarsson,&nbsp;Naser Farrokhi","doi":"10.1002/elps.202400171","DOIUrl":"10.1002/elps.202400171","url":null,"abstract":"<p>Western flower thrips, <i>Frankliniella occidentalis</i> (<i>Thysanoptera</i>: Thripidae) is an invasive agricultural pest with developed resistance to abamectin in some strains due to frequent treatment with the pesticide. In this study, we examined differentially expressed proteins (DEPs) between abamectin-resistant (Aba^R; under abamectin selective pressure) and susceptible strains (Aba^S; without abamectin selective pressure) of <i>F. occidentalis</i>. Proteins were isolated from second instar larvae of both strains and separated via two-dimensional polyacrylamide gel electrophoresis. Nano-flow liquid chromatography–tandem mass spectrometry identified selected protein spot features. From 70 DEPs, 43 spot features were identified: A total of 23 showed an increase in abundance, and 20 were down-regulated in response to abamectin pressure. The enzymatic and structural proteins were classified into the functional groups of macromolecular metabolisms, signaling and cellular processes, immune system, genetic information processing, and exoskeleton-related proteins. The up-regulation of exoskeleton-related proteins may contribute to forming a thicker cuticle, potentially hindering abamectin penetration, which is an interesting finding that needs further investigation. Two novel proteins, triacylglycerol lipase and cuticle protein CPF 2, were only expressed in Aba^R. This work provides insights into abamectin resistance mechanisms in <i>F. occidentalis</i>, which will provide important information for developing insecticide resistance management approaches for this pest.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"46 1-2","pages":"112-126"},"PeriodicalIF":3.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11773298/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142946522","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Editorial Board: Electrophoresis 23–24'24","authors":"","doi":"10.1002/elps.202470122","DOIUrl":"https://doi.org/10.1002/elps.202470122","url":null,"abstract":"","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"45 23-24","pages":"2071-2072"},"PeriodicalIF":3.0,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elps.202470122","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143244951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of Hemoglobin Variants by Capillary Electrophoresis, UV-Vis, and FTIR Spectroscopy.","authors":"Julia Werle, Katerina Dunovska, Jakub Podhajsky, Michal Cerny, Jana Cepova, Arli Aditya Parikesit, Geir Bjørklud, Karel Kotaska, Eva Klapkova, Richard Prusa, Egon Werle, Rene Kizek","doi":"10.1002/elps.202400154","DOIUrl":"https://doi.org/10.1002/elps.202400154","url":null,"abstract":"<p><p>Hemoglobinopathies, hereditary disorders affecting the structure or production of hemoglobin, were detected by routine HbA_1c measurements by capillary electrophoresis (CE) at the University Hospital Motol, Prague. The potential of ultraviolet-visible (UV-Vis) and Fourier-transform infrared (FTIR) spectroscopy for the detection and characterization of hemoglobinopathies was investigated. FTIR spectra were recorded with a very high resolution (0.5 cm^-1) with 128 scans. The broad amide I peak, located at 1700-1600 cm^-1, can be formed by superimposition of the conformational structures of hemoglobin. These secondary protein structures were subjected to mathematical analysis. The application of band narrowing techniques, followed by curve fitting and integration processes, provided the basis for the quantitative estimation of protein secondary structure. As a result, unambiguous differences in UV-Vis spectra among patients with presumably normal hemoglobin, an HbC or a hemoglobin S/hemoglobin G (HbS/HbG)-Philadelphia variant could not be demonstrated. However, FTIR spectra indicated slight differences in α-helix, β-turns, β-sheet, or random coil secondary hemoglobin structures for these mutations. In the spectral wavenumber range of 950-850 cm^-1, there were some obvious FTIR differences at specific wavenumbers between patients with normal hemoglobin and those with the HbC variant. Further investigations are needed with a sufficient number of hemoglobin variants to elucidate the potency of FTIR spectroscopy for the characterization of hemoglobinopathies.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142946480","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Contents: Electrophoresis 23–24'24","authors":"","doi":"10.1002/elps.202470123","DOIUrl":"https://doi.org/10.1002/elps.202470123","url":null,"abstract":"","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"45 23-24","pages":"2073-2074"},"PeriodicalIF":3.0,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143244952","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Ge.F.I. Collaborative Study: Evaluating Reproducibility and Accuracy of a DNA-Methylation-Based Age-Predictive Assay for Routine Implementation in Forensic Casework","authors":"Martina Onofri,&nbsp;Federica Alessandrini,&nbsp;Serena Aneli,&nbsp;Loredana Buscemi,&nbsp;Elena Chierto,&nbsp;Matteo Fabbri,&nbsp;Paolo Fattorini,&nbsp;Paolo Garofano,&nbsp;Fabiano Gentile,&nbsp;Silvano Presciuttini,&nbsp;Carlo Previderè,&nbsp;Carlo Robino,&nbsp;Simona Severini,&nbsp;Federica Tommolini,&nbsp;Pamela Tozzo,&nbsp;Andrea Verzeletti,&nbsp;Eugenia Carnevali","doi":"10.1002/elps.202400190","DOIUrl":"10.1002/elps.202400190","url":null,"abstract":"<p>The increasing interest in DNA methylation (DNAm) analysis within the forensic scientific community prompted a collaborative project by Ge.F.I. (Genetisti Forensi Italiani). The study evaluated a standardized bisulfite conversion–based Single Base Extension (SBE) protocol for the analysis of the methylation levels at five age-predictive loci (ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59). The study encompassed three phases: (1) setting up and validating the protocol to ensure consistency and reproducibility; (2) comparing fresh peripheral blood with blood spots; and (3) evaluating sources of intra- and inter-laboratory variability. Samples from 22 Italian volunteers were analyzed by 6 laboratories in replicates for a total of 528 records. From phase I emerged that the choice of genetic sequencer significantly contributed to inter-laboratory data variation, resulting in separate regression analyses performed for each laboratory. In phase II, blood spots were found to be a reliable source for DNAm analysis, despite exhibiting increased experimental variation compared to fresh peripheral blood. In phase III, a strong correlation between the individual's predicted and true ages was observed across different laboratories. Analysis of variance (ANOVA) of the residuals indicated that one-third of the total variance could be attributed to laboratory-specific factors, whereas two-thirds could be attributed to inter-individual biological differences. The leave-one-out cross-validation (LOO-CV) method yielded an overall mean absolute deviation (MAD) value of 4.41 years, with an average 95% confidence interval of 5.24 years. Stepwise regression analysis proved that a restricted model (ELOVL2, C1orf132/MIR29B2C, and TRIM59) produced results virtually indistinguishable from the five-loci model. Additionally, the analysis of samples in replicates greatly improved the fit of the regression model, balancing the slight effects of intra-laboratory variability. In conclusion, the bisulfite conversion–based SBE protocol, combined with replicate analysis and in-lab calibration of a regression-prediction model, proves to be a reliable and easily implementable method for age prediction in forensic laboratories.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"46 1-2","pages":"76-91"},"PeriodicalIF":3.0,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11773317/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143055888","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isoelectric Focusing Fractionation Method for Signal Enhancement in Detection of Inactivated Biological Agents Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry","authors":"Filip Duša,&nbsp;Jiří Šalplachta,&nbsp;Marie Horká,&nbsp;Kamila Lunerová,&nbsp;Veronika Čermáková,&nbsp;Michal Dřevínek,&nbsp;Oldřich Kubíček","doi":"10.1002/elps.202400052","DOIUrl":"10.1002/elps.202400052","url":null,"abstract":"<p>Timely identification of highly pathogenic bacteria is crucial for efficient mitigation of the connected harmful health effects. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of intact cells enables fast identification of the microorganisms based on their mass spectrometry protein fingerprint profiles. However, the MALDI-TOF MS examination must be preceded by a time-demanding cultivation of the native bacteria to isolate representative cell samples to obtain indicative fingerprints. Isoelectric focusing (IEF) is capable of separating bacterial cells according to their isoelectric point while effectively removing other non-focusing compounds from sample matrix. In this work, we present a divergent-flow IEF chip (DF-IEF chip) fractionation as an alternative way for sample clean-up and concentration of bacterial cells to prepare samples usable for following MALDI-TOF MS analysis without the need of time-demanding cultivation. By means of DF-IEF chip method, we processed four species of highly pathogenic bacteria (<i>Bacillus anthracis</i>, <i>Brucella abortus</i>, <i>Burkholderia mallei</i>, and <i>Yersinia pestis</i>) inactivated with H₂O₂ vapors or by heat treatment at 62.5°C for 24 h. The DF-IEF chip method continually separated and concentrated the inactivated bacterial cells for subsequent detection using MALDI-TOF MS. The content of the inactivated bacteria in the DF-IEF chip fractions was evaluated with the MS analysis, where inactivated <i>Y. pestis</i> was found to be the most efficiently focusing species. Sensitivity analysis showed limits as low as 2 × 10⁵ colony forming units per mL for inactivated <i>B. anthracis</i>.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"46 3-4","pages":"212-220"},"PeriodicalIF":3.0,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elps.202400052","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142921174","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"X-phoresis in Micro/Nano Fluidics 2024","authors":"Xiangchun Xuan,&nbsp;Chun Yang","doi":"10.1002/elps.202470121","DOIUrl":"10.1002/elps.202470121","url":null,"abstract":"","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"45 23-24","pages":"2075"},"PeriodicalIF":3.0,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142920629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}