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.

为了发展对复杂的细胞外基质（ECM）组合物对细胞对这些前再生生物支架的反应的影响的更深了解，人们对先进的蛋白质组学技术在表征脱细胞组织中的应用非常感兴趣。然而，样品中胶原蛋白的高丰度阻碍了ECM衍生生物支架中蛋白质的鉴定，这会干扰低丰度成分的检测，而低丰度成分可能是细胞功能的重要调节剂。为了解决这一局限性，我们开发了一种新型的多酶消化方法，该方法使用了源自溶组织梭状芽胞杆菌的高度纯化的胶原酶进行处理。从ECM衍生的蛋白质提取物中选择性去除胶原蛋白，将其相对丰度从最高90％降低至10％以下。此外，我们应用了这种新方法来表征人脱细胞脂肪组织（DAT），人脱细胞松质骨（DCB）和市售牛腱胶原（BTC）的蛋白质组。我们成功地通过所有三种来源证明了胶原酶处理增加了检测深度，并使得能够通过胰蛋白酶/ LysC在标准消化方案中鉴定被胶原掩盖的多种信号蛋白，从而将DAT中鉴定出的蛋白质数量增加了约〜。 2.2倍，DCB约1.3倍，BTC约1.6倍。此外，使用无标记定量的蛋白质组学研究表明DAT和DCB提取物在成分上是截然不同的，并鉴定了许多脂肪生成和成骨蛋白，它们分别在DAT和DCB中始终较高地表达。总体而言，我们已经开发出一种新的加工方法，可用于先进的质谱研究中，以改善源自哺乳动物组织的生物支架的高通量蛋白质组学表征。此外，我们的研究为作为再生医学的细胞指导平台的两个人类脱细胞组织的复杂ECM组成提供了新的见识。我们已经开发出一种新的加工方法，可用于高级质谱研究中，以改善源自哺乳动物组织的生物支架的高通量蛋白质组学表征。此外，我们的研究为作为再生医学的细胞指导平台的两个人类脱细胞组织的复杂ECM组成提供了新的见识。我们已经开发出一种新的加工方法，可用于高级质谱研究中，以改善源自哺乳动物组织的生物支架的高通量蛋白质组学表征。此外，我们的研究为作为再生医学的细胞指导平台的两个人类脱细胞组织的复杂ECM组成提供了新的见识。