MALDI-TOF mass spectrometry imaging (MSI) is a powerful tool for studying biomolecule localization in tissue. Protein distributions in tissue provide important histological information; however, large proteins exhibit a high limit of detection in MALDI-MS when compared to their corresponding smaller proteolytic peptides. As a result, several techniques have emerged to digest proteins into more detectable peptides for imaging. Digestion is typically accomplished through trypsin deposition on the tissue, but this technique increases the complexity of the tissue microenvironment, which can limit the number of detectable species. This proof-of-principle study explores tryptic tissue digestion during electroblotting through a trypsin-containing membrane. This approach actively extracts and enzymatically digests proteins from mouse brain tissue sections while simultaneously reducing the complexity of the tissue microenvironment (compared to trypsin deposition on the surface) to obtain an increased number of detectable peptide fragments. The method does not greatly compromise spatial location or require expensive devices to uniformly deposit trypsin on tissue. Using electrodigestion through membranes, we detected and tentatively identified several tryptic peptides that were not observed after on-tissue digestion. Moreover, the use of pepsin rather than trypsin in digestion membranes allows extraction and digestion at low pH to detect peptides from a complementary subset of tissue proteins. Future studies will aim to further improve the method, including changing the substrate membrane to increase spatial resolution and the number of detected peptides.

中文翻译：

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通过酶膜进行电印迹以增强分子组织成像。


MALDI-TOF 质谱成像 (MSI) 是研究组织中生物分子定位的强大工具。组织中的蛋白质分布提供了重要的组织学信息；然而，与相应的较小蛋白水解肽相比，大蛋白在 MALDI-MS 中表现出较高的检测限。因此，出现了几种将蛋白质消化成更可检测的肽以进行成像的技术。消化通常是通过胰蛋白酶沉积在组织上来完成的，但这种技术增加了组织微环境的复杂性，从而限制了可检测物种的数量。这项原理验证研究探讨了通过含胰蛋白酶的膜进行电印迹过程中胰蛋白酶组织的消化。这种方法主动从小鼠脑组织切片中提取并酶消化蛋白质，同时降低组织微环境的复杂性（与表面上的胰蛋白酶沉积相比），以获得更多数量的可检测肽片段。该方法不会极大地影响空间位置或需要昂贵的设备来将胰蛋白酶均匀沉积在组织上。使用膜电消化，我们检测并初步鉴定了几种在组织消化后未观察到的胰蛋白酶肽。此外，在消化膜中使用胃蛋白酶而不是胰蛋白酶可以在低 pH 值下进行提取和消化，以检测来自组织蛋白互补子集的肽。未来的研究将旨在进一步改进该方法，包括改变基质膜以提高空间分辨率和检测到的肽的数量。