Lee Su Jin, Adler Belinda, Ekström Simon, Rezeli Melinda, Végvári Ákos, Park Jee-Woong, Malm Johan, Laurell Thomas
Department of Biomedical Engineering, Lund University , P.O. Box 118, SE-211 00 Lund, Sweden.
Anal Chem. 2014 Aug 5;86(15):7627-34. doi: 10.1021/ac501488b. Epub 2014 Jul 11.
With the rapid progress in the development of new clinical biomarkers there is an unmet need of fast and sensitive multiplex analysis methods for disease specific protein monitoring. Immunoaffinity extraction integrated with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis offers a route to rapid and sensitive protein analysis and potentially multiplex biomarker analysis. In this study, the previously reported integrated selective enrichment target (ISET)-MALDI-MS analysis was implemented with ssDNA aptamer functionalized microbeads to address the specific capturing of thrombin in complex samples. The main objective for using an aptamer as the capturing ligand was to avoid the inherently high background components, which are produced during the digestion step following the target extraction when antibodies are used. By applying a thrombin specific aptamer linked to ISET-MALDI-MS detection, a proof of concept of antibody fragment background reduction in the ISET-MALDI-MS readout is presented. Detection sensitivity was significantly increased compared to the corresponding system based on antibody-specific binding as the aptamer ligand does not induce any interfering background residues from the antibodies. The limit of detection for thrombin was 10 fmol in buffer using the aptamer/ISET-MALDI-MS configuration as confirmed by MS/MS fragmentation. The aptamer/ISET-MALDI-MS platform also displayed a limit of detection of 10 fmol for thrombin in five different human serum samples (1/10 diluted), demonstrating the applicability of the aptamer/ISET-MALDI-MS analysis in clinical samples.
随着新型临床生物标志物开发的快速进展，对于疾病特异性蛋白质监测的快速且灵敏的多重分析方法存在未满足的需求。免疫亲和提取与基质辅助激光解吸/电离质谱（MALDI-MS）分析相结合，为快速且灵敏的蛋白质分析以及潜在的多重生物标志物分析提供了一条途径。在本研究中，使用单链DNA适配体功能化微珠实施了先前报道的集成选择性富集靶标（ISET）-MALDI-MS分析，以解决复杂样品中凝血酶的特异性捕获问题。使用适配体作为捕获配体的主要目的是避免在使用抗体进行靶标提取后的消化步骤中产生的固有高背景成分。通过将凝血酶特异性适配体应用于ISET-MALDI-MS检测，展示了在ISET-MALDI-MS读数中降低抗体片段背景的概念验证。与基于抗体特异性结合的相应系统相比，检测灵敏度显著提高，因为适配体配体不会诱导来自抗体的任何干扰性背景残基。通过MS/MS碎片化确认，在缓冲液中使用适配体/ISET-MALDI-MS配置时，凝血酶的检测限为10 fmol。适配体/ISET-MALDI-MS平台在五种不同的人血清样品（1/10稀释）中对凝血酶的检测限也为10 fmol，证明了适配体/ISET-MALDI-MS分析在临床样品中的适用性。
