In quantitative mass spectrometry imaging (MSI), the gold standard adds a single structural homologue of the target compound at a known concentration to the sample. This internal standard enables to map the detected intensity of the target molecule against an external calibration curve. This approach, however, ignores local noise levels and disproportional ion suppression effects, which might depend on the concentration of the target compound. To overcome these issues, we propose a novel approach that applies several isotopically labeled versions, each at a different concentration, to the sample. This allows creating individual internal calibration curves for every MSI pixel. As proof of principle, we have quantified an endogenous peptide of histone H4 by matrix-assisted laser desorption/ionization-Q-MSI (MALDI-Q-MSI), using a mixture of three isotopically labeled versions. The usage of a fourth label allowed us to compare the gold standard to our multilabel approach. We observed substantial heterogeneity in ion suppression across the tissue, which disclosed itself as varying slopes in the per-pixel regression analyses. These slopes were histology-dependent and differed from each other by up to a factor of 4. The results were validated by liquid chromatography–mass spectrometry (LC-MS), exhibiting a high agreement between LC-MS and MALDI-Q-MSI (Pearson correlation r = 0.87). A comparison between the multilabel and single-label approaches revealed a higher accuracy for the multilabel method when the local target compound concentration differed too much from the concentration of the single label. In conclusion, we show that the multilabel approach provides superior quantitation compared to a single-label approach, in case the target compound is inhomogeneously distributed at a wide concentration range in the tissue.

中文翻译：

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质谱成像中的多标签每像素定量

在定量质谱成像（MSI）中，金标准品将已知浓度的目标化合物的单一结构同系物添加到样品中。该内标能够根据外部校准曲线绘制检测到的目标分子强度。但是，这种方法忽略了局部噪声水平和不成比例的离子抑制效应，这可能取决于目标化合物的浓度。为了克服这些问题，我们提出了一种新颖的方法，该方法将几种同位素标记的版本应用于样品，每种版本的浓度不同。这样可以为每个MSI像素创建单独的内部校准曲线。作为原理上的证明，我们通过基质辅助激光解吸/电离-Q-MSI（MALDI-Q-MSI）定量了组蛋白H4的内源肽，使用三种同位素标记版本的混合物。第四个标签的使用使我们可以将金标准与我们的多标签方法进行比较。我们观察到整个组织在离子抑制方面存在很大的异质性，这在每像素回归分析中显示为变化的斜率。这些斜率取决于组织学，并且彼此之间的差异最大为4。结果通过液相色谱-质谱（LC-MS）进行了验证，表明LC-MS和MALDI-Q-MSI之间存在高度一致性（皮尔逊相关r = 0.87）。多标记方法和单标记方法之间的比较显示，当局部目标化合物浓度与单标记浓度相差太大时，多标记方法的准确性更高。总之，我们证明，如果目标化合物在组织中的宽浓度范围内不均匀分布，则与单标记方法相比，多标记方法可提供更好的定量。