色谱法
串联质谱法
蛋白质组学
胰蛋白酶
基质(化学分析)
肾结石
质谱法
草酸钙
生物化学
化学
医学
尿
内科学
基因
酶
作者
Benjamin K. Canales,Lorraine Anderson,LeeAnn Higgins,Joel W. Slaton,Ken P. Roberts,Nathan Liu,Manoj Monga
出处
期刊:Journal of Endourology
[Mary Ann Liebert]
日期:2008-06-01
卷期号:22 (6): 1161-1168
被引量:50
标识
DOI:10.1089/end.2007.0440
摘要
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™ 1.0 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 α-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.
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