Unraveling the Human Bone Microenvironment beyond the Classical Extracellular Matrix Proteins: A Human Bone Protein Library

A characteristic feature of bone, differentiating it from other connective tissues, is the mineralized extracellular matrix (ECM). Mineral accounts for the majority of the bone tissue volume, being the remainder organic material mostly derived from collagen. This, and the fact that only a limited number of noncollagenous ECM proteins are described, provides a limited view of the bone tissue composition and bone metabolism, the more so considering the increasing understanding of ECM significance for cellular form and function. For this reason, we set out to analyze and extensively characterize the human bone proteome using large-scale mass spectrometry-based methods. Bone samples of four individuals were analyzed identifying 3038 unique proteins. A total of 1213 of these were present in at least 3 out of 4 bone samples. For quantification purposes, we were limited to noncollagenous proteins (NCPs) and we could quantify 1051 NCPs. Most classical bone matrix proteins mentioned in literature were detected but were not among the highly abundant ones. Gene ontology analyses identified high-abundance groups of proteins with a functional link to mineralization and mineral metabolism such as transporters, pyrophosphatase activity, and Ca2+-dependent phospholipid binding proteins. ECM proteins were as well overrepresented together with nucleosome and antioxidant activity proteins, which have not been extensively characterized as being important for bone. In conclusion, our data clearly demonstrates that human bone tissue is a reservoir of a wide variety of proteins. In addition to the classical osteoblast-derived ECM, we have identified many proteins from different sources and of unknown function in bone. Thus, this study represents an informative library of bone proteins forming a source for novel bone formation modulators as well as biomarkers for bone diseases such as osteoporosis.

骨骼之特征，区别于其他结缔组织者，在于其矿化的细胞外基质（ECM）。矿物质构成了骨骼组织体积的大部分，而剩余的有机物质主要源自胶原蛋白。加之仅对少量非胶原蛋白ECM蛋白进行了描述，这为我们对骨骼组织组成和骨骼代谢的理解提供了有限的视角，尤其是考虑到对ECM在细胞形态与功能重要性认识的不断深化。鉴于此，我们着手利用大规模质谱分析法，对人类骨骼蛋白组进行了分析与广泛表征。分析了四位个体的骨骼样本，共鉴定出3038种独特的蛋白质。其中，1213种至少在四份骨骼样本中的三份中存在。鉴于定量目的，我们仅限于非胶原蛋白（NCPs），并能够量化1051种NCPs。文献中提及的大多数经典骨骼基质蛋白均被检测到，但并非高丰度蛋白。基因本体分析识别出高丰度蛋白组，这些蛋白与矿化及矿物质代谢等功能存在密切联系，例如转运蛋白、焦磷酸酶活性和Ca2+-依赖性磷脂结合蛋白。细胞外基质蛋白亦显著富集，此外还有核小体和抗氧化活性蛋白，这些蛋白尚未被充分表征为对骨骼的重要性。总之，我们的数据明确表明，人类骨骼组织是多种蛋白质的储存库。除了经典的成骨细胞来源的ECM外，我们还鉴定出许多来自不同来源、功能未知的蛋白质。因此，本研究构成了一个关于骨骼蛋白的信息库，为新型骨骼形成调节剂及骨质疏松等骨骼疾病生物标志物的开发提供了资源。