Horneffer V, Forsmann A, Strupat K, Hillenkamp F, Kubitscheck U
Institute of Medical Physics and Biophysics, University of Münster, Germany.
Anal Chem. 2001 Mar 1;73(5):1016-22. doi: 10.1021/ac000499f.
In this study, the incorporation of Texas Red-labeled avidin into crystals of 2,5-dihydroxybenzoic acid (2,5-DHB) and 2,6-DHB (used as matrixes for matrix-assisted laser desorption/ionization (MALDI)) was investigated by fluorescence spectrophotometry and confocal laser scanning microscopy (CLSM). The analyte distribution in crystals, grown slowly under controlled conditions, was compared to the analyte localization in different standard preparations (dried-droplet and thin-layer preparation). Texas Red turned out to be a useful fluorescence label in the acidic environments of typical matrixes. Earlier results by absorption spectrophotometry could be confirmed by fluorescence measurements; 2,5-DHB incorporates the analyte proportionally, while 2,6-DHB excludes the protein from its crystal lattice. It is found that the analyte distribution can be analyzed well in both single crystals and standard preparation, by CLSM using Texas Red-labeled analytes. The present study allows for a conclusive and consistent interpretation of analyte incorporation into MALDI preparations.
在本研究中，通过荧光分光光度法和共聚焦激光扫描显微镜（CLSM）研究了将德克萨斯红标记的抗生物素蛋白掺入2,5-二羟基苯甲酸（2,5-DHB）和2,6-DHB（用作基质辅助激光解吸/电离（MALDI）的基质）晶体中的情况。将在受控条件下缓慢生长的晶体中的分析物分布与不同标准制剂（干滴和薄层制剂）中的分析物定位进行了比较。结果表明，德克萨斯红在典型基质的酸性环境中是一种有用的荧光标记。吸收分光光度法的早期结果可以通过荧光测量得到证实；2,5-DHB按比例掺入分析物，而2,6-DHB将蛋白质排除在其晶格之外。研究发现，使用德克萨斯红标记的分析物，通过CLSM可以在单晶和标准制剂中很好地分析分析物分布。本研究为分析物掺入MALDI制剂提供了结论性和一致性的解释。
