Localized and analyte-specific normalization for imaging-MALDI-MS - towards quantitative imaging of lipids in tissue

近年来，MALDI-MSI已成为一种在成像质谱学中广泛使用的技术，专用平台现已随处可用。对于组织样本的分析，(磷脂)通常是分析物的选择。除了高丰度和较好的电离效率外，它们在新陈代谢中的重要作用使其成为生物医学研究的主要目标。为了揭示特定脂质物种在特定组织中的作用，正确地描述其在样本中的分布是重要的。为此，必须将分子同一性分配给所确定的m/z值，并且必须将相应的信号强度转换为每个像素的材料量。虽然复杂的硬件和软件解决方案允许根据准确的质量识别脂质信号，但MALDI-MSI的定量仍然是一项艰巨的任务。这是由于样品制备和MALDI过程本身固有的一些因素，这些因素对MALDI中的离子产率有很大影响，不同地区的情况可能会有所不同。这极大地阻碍了将信号强度直接转换为组织中潜在的物质数量。在这方面的一个重要现象是在MALDI过程中不同分析物之间的电荷竞争引起的离子抑制效应。在复杂混合物的分析中，特别明显的是，易电离分析物的存在可以显著降低另一种较不容易电离的化合物的信号强度。最重要的是，组织的物理和化学性质，以及基质和制备方法的选择，都会影响分析物提取到所应用的基质层中，因此也会影响所产生的信号强度。拟议的项目将彻底调查和表征离子抑制效应以及在MALDI-MSI条件下从组织中提取分析物。为此，将产生特定组织区域的脂类提取物，并用ESI-MS进行定量表征。这样，MALDI-MSI中的信号强度可以直接与物质的潜在数量进行比较。在第二种方法中，可以构建和检查具有明确定义的脂质含量的人造组织。这些高度简化的样品允许系统地和定量地分析不同类脂之间的离子抑制效应。在广泛收集数据的基础上，我们将制定个性化的标准化战略。这些将包括使用适合于离子抑制效应的正常化的外部非内源性脂质标准，以及考虑到从组织中提取的位置和分析物特定的校正因子。因此，我们希望提供一种稳健和实用的方法，使MALDI-MSI数据能够直接转换为所分析组织中相对数量的材料的可视化。

MALDI-MSI has in recent years become a heavily used technique in imaging mass spectrometry with dedicated platforms now readily available. For the analysis of tissue samples, (phospho-)lipids are often the analyte of choice. Next to a high abundance and preferable ionization efficiencies, their vital role in the metabolism makes them a prime target of biomedical research. To unveil the role of a specific lipid species in the context of a specific tissue, it is important to correctly depict its distribution within the sample. In order to do so, a molecular identity has to be assigned to the determined m/z-value and the respective signal intensities have to be translated to quantities of material for each pixel. While sophisticated hard- and software solutions allow for the identification of lipid signals based on accurate mass, quantification in MALDI-MSI remains a difficult task. This is due to a number of factors inherent to sample preparation and the MALDI-process itself, that greatly influence ion yields in MALDI and that may vary for different regions. This greatly hampers a straightforward conversion of signal intensity to the underlying amount of material across the tissue. One important phenomenon in this regards is the ion suppression effect that arises from the competition for charge between different analytes during the MALDI-process. Especially apparent in the analysis of complex mixtures the presence of an easily ionizable analyte can significantly decrease the signal intensity of a second compound that is less susceptible for ionization. On top of that, physical and chemical properties of the tissue, but also the choice of matrix and preparation method influence the extraction of analyte into the applied matrix layer and therefore also the resulting signal intensity. The proposed project will thoroughly investigate and characterize both the ion suppression effect as well as the extraction of analyte from tissue under MALDI-MSI conditions. For that lipid extracts of specific tissue regions will be produced and quantitatively characterized with ESI-MS. This way, signal intensities in MALDI-MSI can be directly compared to the underlying amounts of substance. In a second approach, artificial tissue with well-defined lipid content can be constructed and examined. These highly simplified samples allow for the systematic and quantitative analysis of the ion suppression effect between different lipid classes. Building on the broad basis of collected data we will develop individualized normalization strategies. These will include the use of external non-endogenous lipid standards suitable to normalize for ion suppression effects as well as location and analyte specific correction factors accounting for the extraction from the tissue. As a result we hope to deliver a robust and practical method that enables the direct translation of MALDI-MSI data to the visualization of relative amounts of material within the analyzed tissue.