Enhanced Spatially Resolved Proteomics Using On-Tissue Hydrogel Mediated Protein Digestion
Enhanced Spatially Resolved Proteomics Using On-Tissue Hydrogel Mediated Protein Digestion
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DOI：
10.1021/acs.analchem.6b04395
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发表时间：
2017-03-07
影响因子：
7.4
通讯作者：
Caprioli, Richard M.
中科院分区：
文献类型：
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作者：
Rizzo, David G.;Prentice, Boone M.;Caprioli, Richard M.
从组织标本中鉴定蛋白质是生物学研究的一个具有挑战性的领域。目前的许多鉴定技术在样品制备过程中丧失了一定程度的空间信息。最近，已经开发了水凝胶技术，其在下游蛋白质组学分析之前进行空间定位的蛋白质提取和消化。迄今为止，使用这种方法获得的区域特异性蛋白质鉴定仅限于1-2 mm直径的区域。需要用这种技术靶向较小的细胞群体，需要生产较小直径的水凝胶。在这里，我们展示了水凝胶制造过程，使水凝胶应用程序的直径下降到类似于260 μ m，约1/15的面积以前的方法。研究了水凝胶构建中使用的聚丙烯酰胺百分比以及水凝胶负载的胰蛋白酶浓度等参数，以最大限度地提高水凝胶提取物后续液相色谱串联MS（LC-MS/MS）分析的蛋白质鉴定数量。显示18%聚丙烯酰胺浓度比常规的7.5%聚丙烯酰胺浓度提供更刚性的聚合物网络，并且支持使用小穿孔活组织检查来制造单独的水凝胶。在260 μ m的最小水凝胶直径下，仍然可以鉴定出600多种蛋白质。通过对大鼠小脑组织切片的亚区域的分析证明了这些小水凝胶的效用。虽然从每个水凝胶中鉴定了900多种蛋白质，但大约20%的蛋白质是两个区域中每个区域所特有的，这突出了靶向组织亚型以准确表征组织生物学的重要性。这些新改进的水凝胶方法将使研究人员能够针对更小的生物特征进行强大的空间定位蛋白质组学分析。
The identification of proteins from tissue specimens is a challenging area of biological research. Many current techniques for identification forfeit some level of spatial information during the sample preparation process. Recently, hydrogel technologies have been developed that perform spatially localized protein extraction and digestion prior to downstream proteomic analysis. Regiospecific protein identifications acquired using this approach have thus far been limited to 1-2 mm diameter areas. The need to target smaller populations of cells with this technology necessitates the production of smaller diameter hydrogels. Herein, we demonstrate hydrogel fabrication processes that allow hydrogel applications down to a diameter of similar to 260 mu m, approximately 1/15 of the area of previous approaches. Parameters such as the percent polyacrylamide used in hydrogel construction as well as the concentration of trypsin with which the hydrogel is loaded are investigated to maximize the number of protein identifications from subsequent liquid chromatography tandem MS (LC-MS/MS) analysis of hydrogel extracts. An 18% polyacrylamide concentration is shown to provide for a more rigid polymer network than the conventional 7.5% polyacrylamide concentration and supports the fabrication of individual hydrogels using the small punch biopsies. Over 600 protein identifications are still achieved at the smallest hydrogel diameters of 260 mu m. The utility of these small hydrogels is demonstrated through the analysis of sub regions of a rat cerebellum tissue section. While over 900 protein identifications are made from each hydrogel, approximately 20% of the proteins identified are unique to each of the two regions, highlighting the importance of targeting tissue subtypes to accurately characterize tissue biology. These newly improved methods to the hydrogel process will allow researchers to target smaller biological features for robust spatially localized proteomic analyses.