Institute of Life Sciences, Sant'Anna School of Advanced Studies, Pisa, Italy.
Fondazione Toscana Gabriele Monasterio, Pisa, Italy.
Sci Rep. 2023 Oct 31;13(1):18678. doi: 10.1038/s41598-023-45799-5.
The combination of MALDI mass spectrometry imaging, laser-capture microdissection, and quantitative proteomics allows the identification and characterization of molecularly distinct tissue compartments. Such workflows are typically performed using consecutive tissue sections, and so reliable sectioning and mounting of high-quality tissue sections is a prerequisite of such investigations. Embedding media facilitate the sectioning process but can introduce contaminants which may adversely affect either the mass spectrometry imaging or proteomics analyses. Seven low-temperature embedding media were tested in terms of embedding temperature and cutting performance. The two media that provided the best results (5% gelatin and 2% low-melting point agarose) were compared with non-embedded tissue by both MALDI mass spectrometry imaging of lipids and laser-capture microdissection followed by bottom-up proteomics. Two out of the seven tested media (5% gelatin and 2% low-melting point agarose) provided the best performances on terms of mechanical properties. These media allowed for low-temperature embedding and for the collection of high-quality consecutive sections. Comparisons with non-embedded tissues revealed that both embedding media had no discernable effect on proteomics analysis; 5% gelatin showed a light ion suppression effect in the MALDI mass spectrometry imaging experiments, 2% agarose performed similarly to the non-embedded tissue. 2% low-melting point agarose is proposed for tissue embedding in experiments involving MALDI mass spectrometry imaging of lipids and laser-capture microdissection, proteomics of consecutive tissue sections.
MALDI 质谱成像、激光捕获显微切割和定量蛋白质组学的结合允许鉴定和描述分子上不同的组织隔室。这种工作流程通常使用连续的组织切片进行，因此可靠的切片和高质量组织切片的安装是此类研究的前提。包埋介质有助于切片过程，但可能会引入污染物，从而对质谱成像或蛋白质组学分析产生不利影响。在嵌入温度和切割性能方面测试了七种低温嵌入介质。提供最佳结果的两种介质（5%明胶和 2%低熔点琼脂糖）通过 MALDI 质谱成像对脂质和激光捕获显微切割进行了比较，然后进行了自上而下的蛋白质组学分析。在机械性能方面，七种测试介质中的两种（5%明胶和 2%低熔点琼脂糖）表现最佳。这些介质允许低温嵌入和高质量连续切片的收集。与非嵌入组织的比较表明，两种嵌入介质对蛋白质组学分析均无明显影响；5%明胶在 MALDI 质谱成像实验中表现出轻微的离子抑制效应，2%琼脂糖的性能与非嵌入组织相似。建议在涉及脂质 MALDI 质谱成像和激光捕获显微切割、连续组织切片蛋白质组学的实验中使用 2%低熔点琼脂糖进行组织嵌入。
