Thomas Corinne J, Cleland Timothy P, Zhang Sheng, Gundberg Caren M, Vashishth Deepak
Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12182, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12182, USA.
Cornell University Biotechnology Resource Center, Cornell University, Ithaca, NY 14853, USA.
Anal Biochem. 2017 May 15;525:46-53. doi: 10.1016/j.ab.2017.02.011. Epub 2017 Feb 24.
Osteocalcin is an important extracellular matrix bone protein that contributes to the structural properties of bone through its interactions with hydroxyapatite mineral and with collagen I. This role may be affected by glycation, a labile modification the levels of which has been shown to correlate with bone fragility. Glycation starts with the spontaneous addition of a sugar onto a free amine group on a protein, forming an Amadori product, and then proceeds through several environment-dependent stages resulting in the formation of an advanced glycation end product. Here, we induce the first step of this modification on synthetic osteocalcin, and then use multiple mass spectrometry fragmentation techniques to determine the location of this modification. Collision-induced dissociation resulted in spectra dominated by neutral loss, and was unable to identify Amadori products. Electron-transfer dissociation showed that the Amadori product formed solely on osteocalcin's N-terminus. This suggests that the glycation of osteocalcin is unlikely to interfere with osteocalcin's interaction with hydroxyapatite. Instead, glycation may interfere with its interaction with collagen I or another bone protein, osteopontin. Potentially, the levels of glycated osteocalcin fragments released from bone during bone resorption could be used to assess bone quality, should the N-terminal fragments be targeted.
骨钙素是一种重要的细胞外基质骨蛋白，它通过与羟基磷灰石矿物质和I型胶原蛋白相互作用，对骨骼的结构特性产生影响。这种作用可能会受到糖基化的影响，糖基化是一种不稳定的修饰，其水平已被证明与骨脆性相关。糖基化始于糖自发添加到蛋白质上的游离胺基上，形成阿马多里产物，然后经过几个依赖环境的阶段，最终形成晚期糖基化终产物。在此，我们在合成骨钙素上诱导这种修饰的第一步，然后使用多种质谱裂解技术来确定这种修饰的位置。碰撞诱导解离产生的光谱以中性丢失为主，无法识别阿马多里产物。电子转移解离表明，阿马多里产物仅在骨钙素的N端形成。这表明骨钙素的糖基化不太可能干扰骨钙素与羟基磷灰石的相互作用。相反，糖基化可能会干扰其与I型胶原蛋白或另一种骨蛋白骨桥蛋白的相互作用。如果以N端片段为目标，那么在骨吸收过程中从骨骼中释放的糖基化骨钙素片段的水平可能可用于评估骨质量。
