Chemical Digestion-Assisted Proteomics Reveals the Extracellular Matrix Profile of Human Periodontal Ligament and its Alterations in Cultured Cell-Derived Extracellular Matrix

The extracellular matrix (ECM) plays a crucial role in tissue structure and function and serves as an integral component of diverse biological systems. However, the comprehensive characterization of ECM components is challenging because of the insoluble nature of highly cross-linked and glycosylated ECM proteins. This study introduces a chemical digestion-assisted proteomic approach to overcome challenges in ECM profiling, offering an in-depth analysis of the human periodontal ligament (PDL), a tissue critical for oral function and regeneration. Furthermore, we investigated alterations in the ECM composition of cultured PDL cells to provide insights relevant to tissue engineering applications. Our protocol combined chemical digestion and deglycosylation to enhance the identification of ECM proteins. Chemical digestion by hydroxylamine improved protein extraction efficiency by approximately two-fold compared to conventional chaotropic extraction, and deglycosylation increased the number of identified ECM proteins without substantially altering the ECM profile, offering robust quantification of ECM components. A sequential protein extraction approach revealed that proteins insoluble by conventional methods are primarily composed of highly cross-linked fibrillar collagen. Through the application of this technique to human PDL tissue, we present a comprehensive ECM profile for the first time, revealing a high collagen content (>80%) and identifying dominant non-collagenous ECM proteins, such as periostin, dermatopontin, and lumican. Our findings highlight the significant differences between the native ECM of PDL tissue and that produced by cultured PDL cells, emphasizing the importance of considering these variations in regenerative strategies. This study offers a robust tool for analyzing the ECM and its dynamics across diverse tissues and under various physiological and pathological conditions. The results enhance our understanding of periodontal tissue and will inform future approaches for periodontal tissue regeneration.