Assessing the reproducibility of labelled antibody binding in quantitative multiplexed immuno-mass spectrometry imaging.
Assessing the reproducibility of labelled antibody binding in quantitative multiplexed immuno-mass spectrometry imaging.
复制标题
定量多路免疫质谱成像中标记抗体结合的可重复性评估。
DOI：
10.1007/s00216-021-03536-9
复制
发表时间：
2021-09
影响因子：
4.3
通讯作者：
Bishop DP
中科院分区：
文献类型：
--
作者：
Mello MG;Westerhausen MT;Singh P;Doble PA;Wanagat J;Bishop DP
免疫-质谱仪成像(IMSI)使用激光消融-电感耦合等离子体质谱(LA-ICP-MS)来确定免疫标记后生物分子在组织切片中的空间表达。与所有免疫标记技术一样，多重染色的结合效率可能会受到许多因素的影响，包括表位阻断和其他形式的空间位阻。到目前为止，在多重分析中，对金属结合抗体与其表位结合的影响还有待于IMSI的定量研究。在这里，我们描述了一种方法，以肌肉蛋白、肌营养不良蛋白、蔗糖蛋白和肌球蛋白为模型，在将抗体与Maxpar®试剂偶联后，使用标准免疫标记方案和LA-ICPMS成像技术，研究多路传输对可重复结合的影响。每个抗体分别应用于八个部分，并多路传输到另外八个部分。在统计分析发现单独使用抗体和多路应用抗体之间没有显著差异之前，测定了稀土分析物的平均浓度。这些分析为确保多重IMSI期间抗体结合的重复性提供了一个框架，这将允许对蛋白质-蛋白质相互作用的定量探索，并提供对健康和疾病状态下基本生物过程的更好理解。
Immuno-mass spectrometry imaging (iMSI) uses laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to determine the spatial expression of biomolecules in tissue sections following immunolabelling with antibodies conjugated to a metal reporter. As with all immunolabelling techniques, the binding efficiency of multiplexed staining can be affected by a number of factors including epitope blocking and other forms of steric hindrance. To date, the effects on the binding of metal-conjugated antibodies to their epitopes in a multiplexed analysis have yet to be quantitatively explored by iMSI. Here we describe a protocol to investigate the effects of multiplexing on reproducible binding using the muscle proteins, dystrophin, sarcospan and myosin as a model, with antibodies conjugated with Maxpar® reagents before histological application to murine quadriceps sections using standard immunolabelling protocols and imaging with LA-ICP-MS. The antibodies were each individually applied to eight sections, and multiplexed to another eight sections. The average concentration of the lanthanide analytes was determined, before statistical analyses found there was no significant difference between the individual and multiplexed application of the antibodies. These analyses provide a framework for ensuring reproducibility of antibody binding during multiplexed iMSI, which will allow quantitative exploration of protein-protein interactions and provide a greater understanding of fundamental biological processes during healthy and diseased states.
登录
查看更多内容
影响因子：
4.9
作者：
Marshall JL;Crosbie-Watson RH
通讯作者：
Crosbie-Watson RH
影响因子：
4.6
作者：
Warren C;McDonald D;Capaldi R;Deehan D;Taylor RW;Filby A;Turnbull DM;Lawless C;Vincent AE
通讯作者：
Vincent AE
影响因子：
10.9
作者：
Taube, Janis M.;Akturk, Guray;Bifulco, Carlo B.
通讯作者：
Bifulco, Carlo B.
影响因子：
48
作者：
Giesen, Charlotte;Wang, Hao A. O.;Bodenmiller, Bernd
通讯作者：
Bodenmiller, Bernd
影响因子：
6.1
作者：
Lores-Padin, Ana;Fernandez, Beatriz;Pereiro, Rosario
通讯作者：
Pereiro, Rosario