Urology Unit, Department of Surgery, Flinders Medical Centre and Flinders Clinical and Molecular Medicine, School of Medicine, Flinders University, Bedford Park, South Australia, Australia.
J Proteome Res. 2010 Oct 1;9(10):5402-12. doi: 10.1021/pr1006312.
The aim of this study was to compare the intracrystalline protein profiles of hydroxyapatite (HA), brushite (BR), and uric acid (UA) crystals precipitated from the same urine samples. HA, BR, and UA crystals were precipitated on two different occasions from the same pooled healthy urine. Crystals were washed to remove surface-bound proteins, and their composition was confirmed using Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) coupled with energy dispersive X-ray analysis (EDAX). SDS-PAGE was used for visual comparison of the protein content of the demineralised crystal extracts, which were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). HA comprised nanosized particles interspersed with organic material, which was absent from the BR and UA crystals. The number and type of individual proteins differed between the 3 minerals: 45 proteins were detected in the HA crystal extracts and 77 in the BR crystals, including a number of keratins, which were regarded as methodological contaminants. After excluding the keratins, 21 proteins were common to both HA and BR crystals. Seven nonkeratin proteins were identified in the UA extracts. Several proteins consistently detected in the HA and BR crystal extracts have been previously implicated in kidney stone disease, including osteopontin, prothrombin, protein S100A9 (calgranulin B), inter-α-inhibitor, α1-microglobulin bikunin (AMBP), heparan sulfate proteoglycan, and Tamm-Horsfall glycoprotein, all of which are strong calcium binders. We concluded that the association of proteins with HA, BR, and UA crystals formed in healthy urine is selective and that only a few of the numerous proteins present in healthy urine are likely to play any significant role in preventing stone pathogenesis.
本研究旨在比较从同一尿液样本中沉淀的羟基磷灰石（HA）、二水草酸钙（BR）和尿酸（UA）晶体的晶内蛋白谱。将 HA、BR 和 UA 晶体从同一混合健康尿液中两次沉淀。用傅里叶变换红外光谱（FTIR）和场发射扫描电子显微镜（FESEM）结合能谱（EDAX）对晶体进行分析，以确认晶体的组成。用 SDS-PAGE 对去矿化晶体提取物中的蛋白含量进行直观比较，并用液相色谱-串联质谱（LC-MS/MS）对其进行分析。HA 由纳米级颗粒和有机物质组成，而 BR 和 UA 晶体中则没有这些物质。3 种矿物质的个别蛋白数量和类型不同：在 HA 晶体提取物中检测到 45 种蛋白，BR 晶体中检测到 77 种蛋白，其中包括一些角蛋白，被认为是方法学污染物。排除角蛋白后，HA 和 BR 晶体共有 21 种蛋白。UA 提取物中鉴定出 7 种非角蛋白蛋白。在 HA 和 BR 晶体提取物中一致检测到的几种蛋白以前与肾结石病有关，包括骨桥蛋白、凝血酶原、S100A9 蛋白（钙粒蛋白 B）、α1-抑制物、α1-微球蛋白结合素（AMBP）、硫酸乙酰肝素蛋白聚糖和 Tamm-Horsfall 糖蛋白，它们都是强钙结合蛋白。我们得出结论，蛋白质与在健康尿液中形成的 HA、BR 和 UA 晶体的结合是有选择性的，只有少数存在于健康尿液中的众多蛋白质可能在预防结石发病机制方面发挥重要作用。
