Comprehensive proteomic analysis of human calcium oxalate monohydrate kidney stone matrix
Comprehensive proteomic analysis of human calcium oxalate monohydrate kidney stone matrix
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DOI：
10.1089/end.2007.0440
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发表时间：
2008-06-01
影响因子：
2.7
通讯作者：
Monga, Manoj
中科院分区：
文献类型：
--
作者：
Canales, Benjamin K.;Anderson, Lorraine;Monga, Manoj
背景和目的：以前鉴定结石基质蛋白质含量的努力受到缺乏分析高度不溶性蛋白质-晶体复合物所需技术的限制。我们的研究目的是表征基质的草酸钙一水合物（COM）结石使用一个全面的蛋白质组学approach.Materials和方法：七个纯COM石头粉末，蛋白质提取使用四种不同的缓冲溶液。在胰蛋白酶消化之前，使用洗涤剂净化旋转柱或浓缩器去除洗涤剂并交换缓冲液。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。使用ProteinPilot（TM）软件（Applied Biosystems，Inc.）用于蛋白质命中;结果：在4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%的置信度鉴定了68种不同的蛋白质。超过50种蛋白质以前在结石基质中未被鉴定。特别值得注意的是，一个显着数量的炎症蛋白进行了鉴定，包括免疫球蛋白，防御素α-3，clusterin，补体C3 a，激肽原，和fibrinogen.Conclusions：SDS还原缓冲液是有效的溶解蛋白质从石基质进一步MS为基础的蛋白质组学分析。多种细胞蛋白、结构蛋白和血浆蛋白构成COM结石基质。几种石蛋白参与细胞损伤途径，这表明炎症在人类COM石形成中发挥作用。
Background and Purpose: 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.Materials and Methods: 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 (TM) software ( Applied Biosystems, Inc.) for protein hits; peptide MS/MS spectra were manually inspected.Results: 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 alpha-3, clusterin, complement C3a, kininogen, and fibrinogen.Conclusions: 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.