Fluorescein-based low-molecular-mass markers of isoelectric point for tracing pH gradient using high sensitivity capillary isoelectric focusing with laser-induced fluorescence detection
Pavlína Dadajová, Richard Čmelík, Karel Šlais, Filip Duša
{"title":"Fluorescein-based low-molecular-mass markers of isoelectric point for tracing pH gradient using high sensitivity capillary isoelectric focusing with laser-induced fluorescence detection","authors":"Pavlína Dadajová, Richard Čmelík, Karel Šlais, Filip Duša","doi":"10.1016/j.aca.2024.343331","DOIUrl":null,"url":null,"abstract":"<h3>Background</h3>Successful implementation of capillary isoelectric focusing (cIEF) in general requires the use of reference points - markers of isoelectric point. The low-molecular-mass fluorescent compounds based on the fluorescein structure, capable of marking distinct isoelectric points (p<em>I</em>s) with good focusing ability, stability, high extinction coefficient, and fluorescence are an alternative to the peptide and protein p<em>I</em> markers.<h3>Results</h3>Four synthetic pathways were used to produce the ampholytic compounds and the structures along with their basic physical and chemical properties are given. Previously published low-molecular-mass p<em>I</em> markers were used to calibrate the candidate fluorescent compounds using capillary IEF (cIEF) with laser-induced fluorescence (LIF) detection. Linear and point-to-point p<em>I</em> calibration approach were compared for the p<em>I</em> evaluation. The results show 21 fluorescent p<em>I</em> markers (FPIMs). This group of markers covers pH range from 3.10 to 10.21. The usability of the developed FPIMs was further demonstrated by analysis of fluorescein isothiocyanate (FITC) labeled immunoglobin (IgG) standard.<h3>Significance and Novelty</h3>p<em>I</em> markers are crucial for tracking the pH gradient and p<em>I</em> determination of biological ampholytic compounds. This paper presents and characterizes a novel p<em>I</em> marker set capable of tracing isoelectric points and supporting highly sensitive LIF detection-based applications. A basis of a newly developed cIEF-based fractionation method is described as well.","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":null,"pages":null},"PeriodicalIF":5.7000,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytica Chimica Acta","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1016/j.aca.2024.343331","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
引用次数: 0
Abstract
Background
Successful implementation of capillary isoelectric focusing (cIEF) in general requires the use of reference points - markers of isoelectric point. The low-molecular-mass fluorescent compounds based on the fluorescein structure, capable of marking distinct isoelectric points (pIs) with good focusing ability, stability, high extinction coefficient, and fluorescence are an alternative to the peptide and protein pI markers.
Results
Four synthetic pathways were used to produce the ampholytic compounds and the structures along with their basic physical and chemical properties are given. Previously published low-molecular-mass pI markers were used to calibrate the candidate fluorescent compounds using capillary IEF (cIEF) with laser-induced fluorescence (LIF) detection. Linear and point-to-point pI calibration approach were compared for the pI evaluation. The results show 21 fluorescent pI markers (FPIMs). This group of markers covers pH range from 3.10 to 10.21. The usability of the developed FPIMs was further demonstrated by analysis of fluorescein isothiocyanate (FITC) labeled immunoglobin (IgG) standard.
Significance and Novelty
pI markers are crucial for tracking the pH gradient and pI determination of biological ampholytic compounds. This paper presents and characterizes a novel pI marker set capable of tracing isoelectric points and supporting highly sensitive LIF detection-based applications. A basis of a newly developed cIEF-based fractionation method is described as well.
背景毛细管等电聚焦(cIEF)的成功实施一般需要使用参考点--等电点标记物。基于荧光素结构的低分子质量荧光化合物能够标记不同的等电点(pIs),具有良好的聚焦能力、稳定性、高消光系数和荧光性,是肽和蛋白质 pI 标记的替代品。使用毛细管 IEF(cIEF)和激光诱导荧光(LIF)检测法校准了以前发表的低分子质量 pI 标记的候选荧光化合物。对线性和点对点 pI 校准方法进行了比较。结果显示了 21 种荧光 pI 标记(FPIMs)。这组标记的 pH 值范围从 3.10 到 10.21。通过分析异硫氰酸荧光素(FITC)标记的免疫球蛋白(IgG)标准,进一步证明了所开发的 FPIMs 的可用性。本文介绍了一种新型 pI 标记集,它能够追踪等电点并支持基于 LIF 检测的高灵敏度应用。本文还介绍了新开发的基于 cIEF 的分馏方法的基础。
期刊介绍:
Analytica Chimica Acta has an open access mirror journal Analytica Chimica Acta: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review.
Analytica Chimica Acta provides a forum for the rapid publication of original research, and critical, comprehensive reviews dealing with all aspects of fundamental and applied modern analytical chemistry. The journal welcomes the submission of research papers which report studies concerning the development of new and significant analytical methodologies. In determining the suitability of submitted articles for publication, particular scrutiny will be placed on the degree of novelty and impact of the research and the extent to which it adds to the existing body of knowledge in analytical chemistry.