Multiplex bioimaging of proteins-related to neurodegenerative diseases in eye sections by laser ablation - Inductively coupled plasma - Mass spectrometry using metal nanoclusters as labels
Multiplex bioimaging of proteins-related to neurodegenerative diseases in eye sections by laser ablation - Inductively coupled plasma - Mass spectrometry using metal nanoclusters as labels
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DOI：
10.1016/j.talanta.2020.121489
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发表时间：
2021-01-01
期刊：
影响因子：
6.1
通讯作者：
Pereiro, Rosario
中科院分区：
文献类型：
--
作者：
Lores-Padin, Ana;Fernandez, Beatriz;Pereiro, Rosario
同时测定蛋白质与微米分辨率是一个重大的挑战。在这项研究中，采用激光消融（LA）电感耦合等离子体质谱（ICP-MS），使用三种不同的金属纳米簇（MNC）标记的抗体来定量与眼部疾病年龄相关性黄斑变性（AMD）相关的蛋白质的分布。PtNCs、AuNCs和AgNCs含有数百个金属原子，并用于检测来自人尸体眼切片的视网膜、睫状体、视网膜色素上皮（RPE）、脉络膜和巩膜中的金属硫蛋白1/2（MT 1/2）、补体因子H（CFH）和淀粉样前体蛋白（APP）。首先，根据免疫标记方案优化MNC生物缀合的一抗（Ab）的标记，以避免MNC与组织的非特异性相互作用。然后，研究LA和ICP-MS条件，以获得高分辨率图像，用于在同一组织切片上同时检测三种标记。当使用分别为310、723和1194的AuNCs、AgNCs和PtNCs标记的Ab时，发现显著的信号放大。在对MNCs标记的免疫探针进行表征后，使用Ab标记来测定RPE-脉络膜-巩膜中的MT 1/2、CFH和APP，其中观察到与AMD相关的细胞外沉积物的积累。实验结果表明，该方法完全适合于同时检测至少三种不同的蛋白质。
Simultaneous determination of proteins with micrometric resolution is a significant challenge. In this study, laser ablation (LA) inductively coupled plasma - mass spectrometry (ICP-MS) was employed to quantify the distribution of proteins associated to the eye disease age-related macular degeneration (AMD) using antibodies labelled with three different metal nanoclusters (MNCs). PtNCs, AuNCs and AgNCs contain hundreds of metal atoms and were used to detect metallothionein 1/2 (MT1/2), complement factor H (CFH) and amyloid precursor protein (APP) in retina, ciliary body, retinal pigment epithelium (RPE), choroid and sclera from human cadaveric eye sections. First, the labelling of MNCs bioconjugated primary antibodies (Ab) was optimised following an immunolabelling protocol to avoid the non-specific interaction of MNCs with the tissue. Then, the LA and ICP-MS conditions were studied to obtain high-resolution images for the simultaneous detection of the three labels at the same tissue section. A significant signal amplification was found when using AuNCs, AgNCs and PtNCs labelled Ab of 310, 723 and 1194 respectively. After the characterisation of MNCs labelled immunoprobes, the Ab labelling was used for determination of MT1/2, CFH and APP in the RPE-choroid-sclera, where accumulation of extracellular deposits related to AMD was observed. Experimental results suggest that this method is fully suitable for the simultaneous detection of at least three different proteins.