Dufresne Martin, Migas Lukasz G, Djambazova Katerina V, Colley Madeline E, Van de Plas Raf, Spraggins Jeffrey M
Mass Spectrometry Research Center, Vanderbilt University, Nashville, TN, USA; Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA.
Delft Center for Systems and Control, Delft University of Technology, Delft, Netherlands.
Anal Chim Acta. 2025 Oct 15;1371:344423. doi: 10.1016/j.aca.2025.344423. Epub 2025 Jul 15.
We have developed a new class of dual polarity molecules for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) capable of acquiring 5 μm pixel sizes with high sensitivity toward polar lipids and metabolites. Aminated cinnamic acid analogs (ACAAs) are vacuum stable, have high extinction coefficients at 355 nm, are highly sensitive to polar lipids, have low toxicity, and are affordable. Current molecules used for high spatial resolution MALDI IMS of polar lipids have shown great success, but are plagued with issues such as low sensitivity at high spatial resolution, vacuum instability, and/or high toxicity.
ACAAs were evaluated as MALDI matrices, testing them for vacuum stability, absorption at 355 nm, crystal size, sensitivity, and molecular coverage. Among them, 4-aminocinnamic acid (ACA) and 4-(dimethylamino)cinnamic acid (DMACA) were found to perform better than conventional MALDI matrices for lipid IMS experiments. ACA generated fewer in-source fragments due to its high extinction coefficient at 355 nm. This leads to better discernment of thermally labile molecules such as gangliosides compared to typical 'soft' ionization matrices like DHA using murine brain tissue. On the other hand, DMACA showed better optical properties than ACA, giving it higher sensitivity from many lipid classes, such as phospholipids and sulfatides. DMACA outperformed DAN and DHA at their individually optimized laser power at small pixel sizes (≤10 μm). DMACA also allows for lower laser power to be used without compromising sensitivity, which reduced the laser spot size at the sample surface from ∼6 μm to ∼4.5 μm without hardware modifications.
Improved sensitivity and absorption efficiency at 355 nm allow for 5 μm pixel size MALDI IMS without oversampling while maintaining high S/N on commercial mass spectrometry platforms. Performing MALDI experiments at reduced laser energies minimizes tissue damage, enabling advanced multimodal MALDI IMS studies to be performed on single tissue sections. Comparisons and optimized MALDI IMS methods were performed on murine tissues and human kidney samples as part of the Human Biomolecular Atlas Program.
我们开发了一类新型的双极性分子，用于基质辅助激光解吸/电离（MALDI）成像质谱（IMS），能够以5μm的像素尺寸对极性脂质和代谢物进行高灵敏度检测。氨基肉桂酸类似物（ACAA）在真空中稳定，在355nm处具有高消光系数，对极性脂质高度敏感，毒性低且价格实惠。目前用于极性脂质高空间分辨率MALDI IMS的分子已取得巨大成功，但存在诸如高空间分辨率下灵敏度低、真空不稳定性和/或高毒性等问题。
对ACAA作为MALDI基质进行了评估，测试了它们的真空稳定性、在355nm处的吸收、晶体尺寸、灵敏度和分子覆盖率。其中，发现4-氨基肉桂酸（ACA）和4-（二甲基氨基）肉桂酸（DMACA）在脂质IMS实验中比传统MALDI基质表现更好。由于ACA在355nm处具有高消光系数，其在源内产生的碎片较少。与使用鼠脑组织的典型“软”电离基质（如DHA）相比，这使得对神经节苷脂等热不稳定分子的辨别能力更强。另一方面，DMACA的光学性质比ACA更好，使其对许多脂质类别（如磷脂和硫脂）具有更高的灵敏度。在小像素尺寸（≤10μm）下，DMACA在其各自优化的激光功率下优于DAN和DHA。DMACA还允许在不降低灵敏度的情况下使用更低的激光功率，这在不进行硬件修改的情况下将样品表面的激光光斑尺寸从约6μm减小到约4.5μm。
在355nm处提高的灵敏度和吸收效率使得在不进行过采样的情况下能够实现5μm像素尺寸的MALDI IMS，同时在商业质谱平台上保持高信噪比。在降低的激光能量下进行MALDI实验可将组织损伤降至最低，从而能够在单个组织切片上进行先进的多模态MALDI IMS研究。作为人类生物分子图谱计划的一部分，在鼠组织和人类肾脏样本上进行了比较和优化的MALDI IMS方法。
