Department of Biochemistry, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, N6A 5C1, Canada.
Department of Anatomy and Cell Biology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, N6A 5C1, Canada.
Biomaterials. 2017 Nov;144:130-143. doi: 10.1016/j.biomaterials.2017.08.012. Epub 2017 Aug 13.
There is great interest in the application of advanced proteomic techniques to characterize decellularized tissues in order to develop a deeper understanding of the effects of the complex extracellular matrix (ECM) composition on the cellular response to these pro-regenerative bioscaffolds. However, the identification of proteins in ECM-derived bioscaffolds is hindered by the high abundance of collagen in the samples, which can interfere with the detection of lower-abundance constituents that may be important regulators of cell function. To address this limitation, we developed a novel multi-enzyme digestion approach using treatment with a highly-purified collagenase derived from Clostridium Histolyticum to selectively deplete collagen from ECM-derived protein extracts, reducing its relative abundance from up to 90% to below 10%. Moreover, we applied this new method to characterize the proteome of human decellularized adipose tissue (DAT), human decellularized cancellous bone (DCB), and commercially-available bovine tendon collagen (BTC). We successfully demonstrated with all three sources that collagenase treatment increased the depth of detection and enabled the identification of a variety of signaling proteins that were masked by collagen in standard digestion protocols with trypsin/LysC, increasing the number of proteins identified in the DAT by ∼2.2 fold, the DCB by ∼1.3 fold, and the BTC by ∼1.6 fold. In addition, quantitative proteomics using label-free quantification demonstrated that the DAT and DCB extracts were compositionally distinct, and identified a number of adipogenic and osteogenic proteins that were consistently more highly expressed in the DAT and DCB respectively. Overall, we have developed a new processing method that may be applied in advanced mass spectrometry studies to improve the high-throughput proteomic characterization of bioscaffolds derived from mammalian tissues. Further, our study provides new insight into the complex ECM composition of two human decellularized tissues of interest as cell-instructive platforms for regenerative medicine.
人们对应用先进的蛋白质组学技术来对脱细胞组织进行特征分析很感兴趣，目的是为了更深入地了解复杂细胞外基质（ECM）组成对细胞对这些再生生物支架反应的影响。然而，在 ECM 衍生生物支架中鉴定蛋白质时，由于样本中胶原含量丰富，这会干扰对低丰度成分的检测，而这些低丰度成分可能是细胞功能的重要调节剂。为了解决这个限制，我们开发了一种新的多酶消化方法，使用源自溶组织梭菌的高度纯化胶原酶处理，从 ECM 衍生的蛋白质提取物中选择性地耗尽胶原，将其相对丰度从高达 90%降低到 10%以下。此外，我们应用这种新方法来对人脱细胞脂肪组织（DAT）、人脱细胞松质骨（DCB）和市售牛腱胶原（BTC）的蛋白质组进行特征分析。我们成功地用这三种来源证明，胶原酶处理增加了检测深度，并使各种信号蛋白得以鉴定，这些信号蛋白在标准的胰蛋白酶/赖氨酰肽链内切酶（LysC）消化方案中被胶原掩盖，使 DAT 中鉴定的蛋白质数量增加了约 2.2 倍，DCB 增加了约 1.3 倍，BTC 增加了约 1.6 倍。此外，使用无标记定量法的定量蛋白质组学表明，DAT 和 DCB 提取物在组成上是不同的，并鉴定出了许多在 DAT 和 DCB 中分别高度表达的脂肪形成和成骨蛋白。总的来说，我们开发了一种新的处理方法，可应用于高级质谱研究，以提高对哺乳动物组织衍生生物支架的高通量蛋白质组学特征分析。此外，我们的研究为两种人类脱细胞组织（DAT 和 DCB）的复杂 ECM 组成提供了新的见解，因为它们是再生医学中细胞指令性的平台。
