Dysregulation of cell signaling in chondrocytes and in bone cells, such as osteocytes, osteoblasts, osteoclasts, and an elevated burden of senescent cells in cartilage and bone, are implicated in osteoarthritis (OA). Mass spectrometric analyses provides a crucial molecular tool-kit to understand complex signaling relationships in age-related diseases, such as OA. Here we introduce a novel mass spectrometric workflow to promote proteomic studies of bone and cartilage. This workflow uses highly specialized steps, including extensive overnight demineralization, pulverization, and incubation for 72 h in 6 M guanidine hydrochloride and EDTA, followed by proteolytic digestion. Analysis on a high-resolution Orbitrap Eclipse and Orbitrap Exploris 480 mass spectrometer using Data-Independent Acquisition (DIA) provides deep coverage of the bone proteome, and preserves post-translational modifications, such as hydroxyproline. A spectral library-free quantification strategy, directDIA, identified and quantified over 2,000 protein groups (with ≥ 2 unique peptides) from calcium-rich bone matrices. Key components identified were proteins of the extracellular matrix (ECM), bone-specific proteins (e.g., secreted protein acidic and cysteine rich, SPARC, and bone sialoprotein 2, IBSP), and signaling proteins (e.g., transforming growth factor beta-2, TGFB2), and lysyl oxidase homolog 2 (LOXL2), an important protein in collagen crosslinking. Post-translational modifications (PTMs) were identified without the need for specific enrichment. This includes collagen hydroxyproline modifications, chemical modifications for collagen self-assembly and network formation. Multiple senescence factors were identified, such as complement component 3 (C3) protein of the complement system and many matrix metalloproteinases, that might be monitored during age-related bone disease progression. Our innovative workflow yields in-depth protein coverage and quantification strategies to discover underlying biological mechanisms of bone aging and to provide tools to monitor therapeutic interventions. These novel tools to monitor the bone proteome open novel horizons to investigate bone-specific diseases, many of which are age-related.

中文翻译：

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骨骼蛋白质组的深度覆盖和量化为骨骼生物学和疾病的新发现提供了更多机会

软骨细胞和骨细胞（如骨细胞、成骨细胞、破骨细胞）中细胞信号传导的失调，以及软骨和骨骼中衰老细胞负担的增加，都与骨关节炎 (OA) 有关。质谱分析提供了一个重要的分子工具包，以了解与年龄相关的疾病（如 OA）中复杂的信号传导关系。在这里，我们介绍了一种新颖的质谱工作流程，以促进骨骼和软骨的蛋白质组学研究。此工作流程使用高度专业化的步骤，包括广泛的过夜脱矿质、粉碎和在 6 M 盐酸胍和 EDTA 中孵育 72 小时，然后进行蛋白水解消化。使用数据独立采集 (DIA) 对高分辨率 Orbitrap Eclipse 和 Orbitrap Exploris 480 质谱仪进行的分析提供了对骨骼蛋白质组的深度覆盖，并保留翻译后修饰，例如羟脯氨酸。一种无光谱库的量化策略 directDIA 从富含钙的骨基质中识别和量化了超过 2,000 个蛋白质组（具有≥ 2 个独特的肽）。确定的关键成分是细胞外基质 (ECM) 的蛋白质、骨特异性蛋白质（例如，分泌蛋白酸性和富含半胱氨酸、SPARC 和骨唾液酸蛋白 2、IBSP）和信号蛋白（例如，转化生长因子 beta-2、 TGFB2) 和赖氨酰氧化酶同系物 2 (LOXL2)，胶原蛋白交联中的重要蛋白质。无需特定富集即可识别翻译后修饰 (PTM)。这包括胶原蛋白羟脯氨酸修饰、胶原蛋白自组装和网络形成的化学修饰。确定了多种衰老因素，例如补体系统的补体成分 3 (C3) 蛋白和许多基质金属蛋白酶，可以在与年龄相关的骨病进展过程中对其进行监测。我们创新的工作流程产生了深入的蛋白质覆盖率和量化策略，以发现骨老化的潜在生物学机制，并提供监测治疗干预的工具。这些用于监测骨蛋白质组的新工具为研究骨特异性疾病开辟了新的视野，其中许多疾病与年龄有关。我们创新的工作流程产生了深入的蛋白质覆盖率和量化策略，以发现骨老化的潜在生物学机制，并提供监测治疗干预的工具。这些用于监测骨蛋白质组的新工具为研究骨特异性疾病开辟了新的视野，其中许多疾病与年龄有关。我们创新的工作流程产生了深入的蛋白质覆盖率和量化策略，以发现骨老化的潜在生物学机制，并提供监测治疗干预的工具。这些用于监测骨蛋白质组的新工具为研究骨特异性疾病开辟了新的视野，其中许多疾病与年龄有关。