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Abstract：
Paneth cells are antimicrobial peptide-secreting cells located at the base of the crypts of the small intestine. The proteome of Paneth cells is not well defined because of their coexistence with stem cells, making it difficult to culture Paneth cells alone in vitro. Using a simplified toluidine blue O method for staining mouse intestinal tissue, laser capture microdissection (LCM) to isolate cells from the crypt region, and surfactant-assisted one-pot protein digestion, we identified more than 1300 proteins from crypts equivalent to 18,000 cells. Compared with the proteomes of villi and smooth muscle regions, the crypt proteome is highly enriched in defensins, lysozymes, and other antimicrobial peptides that are characteristic of Paneth cells. The sensitivity of the LCM-based proteomics approach was also assessed using a smaller number of cell equivalent tissues: a comparable proteomic coverage can be achieved with 3600 cells. This work is the first proteomics study of intestinal tissue enriched with Paneth cells. The simplified workflow enables profiling of Paneth cell-associated pathological changes at the proteome level directly from frozen intestinal tissue. It may also be useful for proteomics studies of other spatially resolved cell types from other tissues.
摘要：
潘氏细胞是位于小肠隐窝底部的抗微生物肽分泌细胞。Paneth细胞的蛋白质组由于与干细胞共存而没有很好的定义，很难在体外单独培养潘氏细胞。使用简化的甲苯胺蓝O方法对小鼠肠组织进行染色，激光捕获显微切割（LCM）从隐窝区域分离细胞，和表面活性剂辅助的一锅蛋白质消化，我们从相当于18,000个细胞的隐窝中鉴定出1300多种蛋白质。与绒毛和平滑肌区的蛋白质组相比，隐窝蛋白质组高度富含防御素，溶菌酶，和其他具有潘氏细胞特征的抗菌肽。基于LCM的蛋白质组学方法的灵敏度也使用较少数量的细胞等效组织进行评估:可以用3600个细胞实现相当的蛋白质组覆盖率。这项工作是第一个富含Paneth细胞的肠组织的蛋白质组学研究。简化的工作流程能够直接从冷冻的肠组织在蛋白质组水平上对Paneth细胞相关的病理变化进行分析。它也可用于来自其他组织的其他空间分辨细胞类型的蛋白质组学研究。
