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J Endourol. 2008 Jun;22(6):1161-7. doi: 10.1089/end.2007.0440.

Second prize: Comprehensive proteomic analysis of human calcium oxalate monohydrate kidney stone matrix.

Author information

1
Department of Urologic Surgery, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA. benjamin.canales@urology.ufl.edu

Abstract

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 -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.

PMID:
18484873
DOI:
10.1089/end.2007.0440
[Indexed for MEDLINE]

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