Bell Jonathan M, Yagnik Gargey, Dettori Leonardo G, Carvalho Philip, Wan Zhi, Rothschild Kenneth J, Lim Mark J
AmberGen, Inc., 44 Manning Road, Billerica, Massachusetts 01821, United States.
Department of Physics and Photonics Center, Boston University, Boston, Massachusetts 02215, United States.
J Am Soc Mass Spectrom. 2025 Aug 6;36(8):1621-1640. doi: 10.1021/jasms.5c00057. Epub 2025 Jul 11.
Many fluorescence-based hybridization (FISH) methods have been developed to spatially resolve DNA (genes) and RNA (transcripts) in tissues. Signal amplification is achieved in a variety of ways, including branched DNA (bDNA) methods that create multiple fluorescent probe binding sites on the target nucleic acid. To avoid spectral overlap, high levels of multiplexing are achieved by extensive cycling, using a few nonoverlapping fluorophores per cycle. However, these methods can be slow, cause accumulating tissue damage, and are negatively impacted by autofluorescence. In addition, FISH-based methods alone do not provide a comprehensive multiomic picture of the complex biological contributions from the different molecular species in a tissue, including metabolites, nucleic acids, proteins, and xenobiotics. We report the development of novel photocleavable mass-tagged oligonucleotide probes generated by copper-free Click chemistry for use with amplified and multiplexed MALDI mass spectrometric imaging-based hybridization (MALDI-ISH). These probes were successfully substituted for fluorescent detector probes using RNAscope but required no cycling. We also demonstrate a fully mass spectrometric workflow that enables multiomic imaging of label-free metabolites (lipids) and targeted transcripts from a single Alzheimer's mouse brain tissue section. Furthermore, we demonstrate a triomic workflow where, in addition to label-free lipids, adding MALDI-ISH combined with MALDI-immunohistochemistry (MALDI-IHC) enables imaging of targeted transcripts and proteins on the same tissue section. K-means cluster analysis of multiomic biomarkers reveals spatial correlations of these various molecular species with Alzheimer's plaques.
已经开发了许多基于荧光的杂交（FISH）方法来在空间上解析组织中的DNA（基因）和RNA（转录本）。信号放大通过多种方式实现，包括分支DNA（bDNA）方法，该方法在靶核酸上创建多个荧光探针结合位点。为了避免光谱重叠，通过广泛循环实现了高水平的多重检测，每个循环使用少数不重叠的荧光团。然而，这些方法可能速度慢，会导致组织损伤累积，并且受到自发荧光的负面影响。此外，仅基于FISH的方法不能提供组织中不同分子种类（包括代谢物、核酸、蛋白质和外源性物质）复杂生物学贡献的全面多组学图景。我们报告了通过无铜点击化学产生的新型光可裂解质量标记寡核苷酸探针的开发，用于基于基质辅助激光解吸电离质谱成像的杂交（MALDI-ISH）的扩增和多重检测。这些探针使用RNAscope成功替代了荧光检测探针，但无需循环。我们还展示了一种完全基于质谱的工作流程，能够对来自单个阿尔茨海默病小鼠脑组织切片的无标记代谢物（脂质）和靶向转录本进行多组学成像。此外，我们展示了一种三重组学工作流程，除了无标记脂质外，添加MALDI-ISH与基质辅助激光解吸电离免疫组织化学（MALDI-IHC）相结合，能够在同一组织切片上对靶向转录本和蛋白质进行成像。多组学生物标志物的K均值聚类分析揭示了这些不同分子种类与阿尔茨海默病斑块的空间相关性。
