Canales Benjamin K, Anderson Lorraine, Higgins Leeann, Slaton Joel, Roberts Ken P, Liu Nathan, Monga Manoj
Department of Urologic Surgery, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.
J Endourol. 2008 Jun;22(6):1161-7. doi: 10.1089/end.2007.0440.
Previous efforts to identify the protein content of stone matrix have been limited by the lack of technology necessary to analyze the highly insoluble protein-crystalline complex. Our study objective is to characterize the matrix of calcium oxalate monohydrate (COM) stones using a comprehensive proteomics approach.
Seven pure COM stones were powdered, and proteins were extracted using four different buffer solutions. Detergent cleanup spin columns or concentrators were used to remove detergent and to exchange buffers before trypsin digestion. Tryptic peptides were analyzed with reversed-phase, high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS) using a QSTAR Pulsar i quadrapole time of flight mass spectrometer. Tandem mass spectra were searched against National Center for Biotechnology Information human nonredundant database using ProteinPilot 1.0 software (Applied Biosystems, Inc.) for protein hits; peptide MS/MS spectra were manually inspected.
Of the four buffers, only 2% sodium dodecyl sulfate (SDS) samples had normal HPLC and MS/MS elution patterns. We identified 68 distinct proteins with 95% confidence. More than 50 of the proteins have not been previously identified in stone matrix. Of particular note, a significant number of inflammatory proteins were identified, including immunoglobulins, defensin -3, clusterin, complement C3a, kininogen, and fibrinogen.
SDS reducing buffer was efficient at solubilizing proteins from stone matrix for further MS-based proteomic analysis. A variety of cellular, structural, and plasma proteins comprise COM stone matrix. Several of the stone proteins are involved in cell injury pathways, which suggests that inflammation plays a role in human COM stone formation.
以往确定结石基质蛋白质含量的努力因缺乏分析高度不溶性蛋白质 - 晶体复合物所需的技术而受到限制。我们的研究目的是使用综合蛋白质组学方法对一水草酸钙（COM）结石的基质进行表征。
将七颗纯COM结石磨成粉末，使用四种不同的缓冲溶液提取蛋白质。在胰蛋白酶消化之前，使用去污剂清理旋转柱或浓缩器去除去污剂并更换缓冲液。使用QSTAR Pulsar i四极杆飞行时间质谱仪通过反相高效液相色谱（RP - HPLC）和串联质谱（MS / MS）分析胰蛋白酶肽段。使用ProteinPilot 1.0软件（应用生物系统公司）在国家生物技术信息中心人类非冗余数据库中搜索串联质谱图以查找蛋白质匹配项；手动检查肽段MS / MS谱图。
在四种缓冲液中，只有2％十二烷基硫酸钠（SDS）样品具有正常的HPLC和MS / MS洗脱模式。我们以95％的置信度鉴定出68种不同的蛋白质。其中超过50种蛋白质以前未在结石基质中被鉴定出来。特别值得注意的是，鉴定出了大量炎症蛋白，包括免疫球蛋白、防御素 - 3、簇集蛋白、补体C3a、激肽原和纤维蛋白原。
SDS还原缓冲液能够有效地溶解结石基质中的蛋白质，以便进行基于质谱的进一步蛋白质组学分析。COM结石基质由多种细胞、结构和血浆蛋白组成。几种结石蛋白参与细胞损伤途径，这表明炎症在人类COM结石形成中起作用。
