Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, Aurora, Colorado, USA.
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, Aurora, Colorado, USA; Cancer Center Proteomics Core, School of Medicine, University of Colorado, Aurora, Colorado, USA.
Mol Cell Proteomics. 2021;20:100079. doi: 10.1016/j.mcpro.2021.100079. Epub 2021 Jun 3.
The extracellular matrix is a key component of tissues, yet it is underrepresented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested, and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications.
细胞外基质是组织的关键组成部分，但在蛋白质组学数据集中的代表性不足。由于许多细胞外基质蛋白在传统的蛋白质提取缓冲液中不溶，因此鉴定和评估细胞外基质（ECM）中的蛋白质具有挑战性。在这里，我们在真实条件下对几种主要方法的去细胞化和细胞外基质提取步骤进行了分离和评估。这些结果用于优化两部分细胞外基质提取方法。还测试了大约十几个其他参数，并根据总体细胞外基质覆盖率或特定细胞外基质类别给出了分析建议。与标准的溶液内消化相比，优化后的方法使独特的细胞外基质肽鉴定增加了四倍。
