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


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.
Canales
Benjamin K
BK
Department of Urologic Surgery, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA. benjamin.canales@urology.ufl.edu
Anderson
Lorraine
L
Higgins
Leeann
L
Slaton
Joel
J
Roberts
Ken P
KP
Liu
Nathan
N
Monga
Manoj
M
D001363:Awards and Prizes; D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry; D002021:Buffers; D002129:Calcium Oxalate / Q000032:analysis*; D006801:Humans; D007249:Inflammation; D007669:Kidney Calculi / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics; D012967:Sodium Dodecyl Sulfate; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
D001798:Blood Proteins; D002021:Buffers; D010455:Peptides; D020543:Proteome; D002129:Calcium Oxalate; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin