Materials Research Institute, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
The Jake Gittlen Laboratories for Cancer Research, Penn State College of Medicine, Hershey, Pennsylvania 17033, United States.
J Am Soc Mass Spectrom. 2021 Jan 6;32(1):255-261. doi: 10.1021/jasms.0c00300. Epub 2020 Oct 28.
Variants of the gene, which encodes a putative potassium-dependent sodium-calcium exchanger (NCKX5) that most likely resides in the melanosome or its precursor, affect pigmentation in both humans and zebrafish (). This finding suggests that genetic variations influencing human skin pigmentation alter melanosome biogenesis via ionic changes. Gaining an understanding of how changes in the ionic environment of organelles impact melanosome morphogenesis and pigmentation will require a spatially resolved way to characterize the chemical environment of melanosomes in pigmented tissue such as retinal pigment epithelium (RPE). The imaging mass spectrometry technique most suited for this type of cell and tissue analysis is time-of-flight secondary ion mass spectrometry (ToF-SIMS) because it is able to detect many biochemical species with high sensitivity and with submicron spatial resolution. Here, we describe chemical imaging of the RPE in frozen-hydrated sections of larval zebrafish using cryo-ToF-SIMS. To facilitate the data interpretation, positive and negative polarity ToF-SIMS image data were transformed into a single hyperspectral data set and analyzed using principal component analysis. The combination of a novel protocol and the use of multivariate data analysis allowed us to discover new marker ions that are attributable to leucodopachrome, a metabolite specific to the biosynthesis of eumelanin. The described methodology may be adapted for the investigation of other classes of molecules in frozen tissues from zebrafish and other organisms.
基因的变异体，该基因编码一种假定的钾依赖的钠-钙交换器（NCKX5），最有可能位于黑素体或其前体中，影响人类和斑马鱼的色素沉着（）。这一发现表明，影响人类皮肤色素沉着的遗传变异通过离子变化改变黑素体的发生。了解细胞器离子环境的变化如何影响黑素体形态发生和色素沉着，需要一种空间分辨的方法来描述色素组织（如视网膜色素上皮（RPE））中黑素体的化学环境。最适合这种类型的细胞和组织分析的成像质谱技术是飞行时间二次离子质谱（ToF-SIMS），因为它能够以亚微米空间分辨率和高灵敏度检测许多生化物质。在这里，我们描述了使用冷冻飞行时间二次离子质谱（cryo-ToF-SIMS）对幼虫斑马鱼冷冻水合切片中的 RPE 进行化学成像。为了便于解释数据，正、负极性 ToF-SIMS 图像数据被转换为单个高光谱数据集，并使用主成分分析进行分析。新方案的组合和多元数据分析的使用使我们能够发现新的标记离子，这些离子归因于白细胞多巴醌，这是一种特定于真黑素生物合成的代谢物。所描述的方法可以适用于从斑马鱼和其他生物体的冷冻组织中研究其他类别的分子。
