Mass spectroscopic characteristics of low molecular weight proteins extracted from calcium oxalate stones: preliminary study

It is believed that boundary compositions of matrix proteins might play a role in stone formation; however, few proteomic studies concerning matrix proteins in urinary stones have been conducted. In this study, we extracted low molecular weight proteins from calcium oxalate stones and measured their characteristic patterns by mass spectroscopy. A total of 10 stones were surgically removed from patients with urolithiasis. Proteins were extracted from the stones and identified by one‐dimensional electrophoresis (sodium dodecyl sulfate buffer [SDS]–polyacrylamide gel electrophoresis [SDS‐PAGE]). After in‐gel digest, samples were analyzed by the surface‐enhanced laser desorption ionization‐time of flight (SELDI‐TOF) technique. The peptide sequences were analyzed from the data of mass spectroscopy. Proteins were identified from Database Search (SwissProt Protein Database; Swiss Institute of Bioinformatics; http://www.expasy.org/sprot) on a MASCOT server (Matrix Science Ltd.; http://www.matrixscience.com). A total of three bands of proteins (27, 18, and 14 kDa) were identified from SDS‐PAGE in each stone sample. A database search (SwissProt) on a MASCOT server revealed that the most frequently seen proteins from band 1 (27 kDa) were leukocyte elastase precursor, cathepsin G precursor, azurocidin precursor, and myeloblastin precursor (EC 3.4.21.76) (leukocyte proteinase 3); band 2 (18 kDa) comprised calgranulin B, eosinophil cationic protein precursor, and lysozyme C precursor; band 3 (14 kDa) showed neutrophil defensin 3 precursor, calgranulin A, calgranulin C, and histone H4. The modifications and deamidations found from the mass pattern of these proteins may provide information for the study of matrix proteins. Various lower molecular weight proteins can be extracted from calcium oxalate stones. The characteristic patterns and their functions of those proteins should be further tested to investigate their roles in stone formation. J. Clin. Lab. Anal. 22:77–85, 2008. © 2008 Wiley‐Liss, Inc.

[1]  F. Coe,et al.  Urine protein markers distinguish stone-forming from non-stone-forming relatives of calcium stone formers. , 2006, American journal of physiology. Renal physiology.

[2]  V. Thongboonkerd,et al.  Identification of human urinary trefoil factor 1 as a novel calcium oxalate crystal growth inhibitor. , 2005, The Journal of clinical investigation.

[3]  J. Lieske,et al.  Urinary macromolecular inhibition of crystal adhesion to renal epithelial cells is impaired in male stone formers. , 2005, Kidney international.

[4]  T. Colgan,et al.  A strategy for high‐resolution protein identification in surface‐enhanced laser desorption/ionization mass spectrometry: Calgranulin A and chaperonin 10 as protein markers for endometrial carcinoma , 2005, Proteomics.

[5]  J. Hughes,et al.  Osteopontin and Calcium Stone Formation , 2004, Nephron Physiology.

[6]  D. Desiderio,et al.  Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests , 2004, Journal of the American Society for Mass Spectrometry.

[7]  R. Eeles,et al.  Proteomics and urine analysis: a potential promising new tool in urology , 2004, BJU international.

[8]  D. Kok,et al.  Modulators of urinary stone formation. , 2004, Frontiers in bioscience : a journal and virtual library.

[9]  T. Okita,et al.  Isolation of a Crystal Matrix Protein Associated with Calcium Oxalate Precipitation in Vacuoles of Specialized Cells1 , 2003, Plant Physiology.

[10]  R. Selvam,et al.  Oxalate binding proteins in calcium oxalate nephrolithiasis , 2003, Urological Research.

[11]  Saeed R. Khan,et al.  Expression of osteopontin in rat kidneys: induction during ethylene glycol induced calcium oxalate nephrolithiasis. , 2002, The Journal of urology.

[12]  S. Shapiro,et al.  Neutrophil elastase targets virulence factors of enterobacteria , 2002, Nature.

[13]  R. Selvam,et al.  Increased calcium oxalate crystal nucleation and aggregation by peroxidized protein of human kidney stone matrix and renal cells , 2001, Urological Research.

[14]  K. Kohri,et al.  Distribution of osteopontin and calprotectin as matrix protein in calcium-containing stone , 1999, Urological Research.

[15]  F Gharahdaghi,et al.  Mass spectrometric identification of proteins from silver‐stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity , 1999, Electrophoresis.

[16]  J. Selengut,et al.  Identification of the calcium-binding protein calgranulin in the matrix of struvite stones. , 1994, Journal of endourology.

[17]  M. E. Guerra,et al.  Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes. , 1991, Journal of immunology.

[18]  Veronica van Heyningen,et al.  A clue to the basic defect in cystic fibrosis from cloning the CF antigen gene , 1987, Nature.

[19]  A. Bhown,et al.  Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil cathepsin G from normal donors. , 1985, Analytical biochemistry.

[20]  G. H. Nancollas.  Biological Mineralization and Demineralization , 1982, Dahlem Workshop Reports.

[21]  E. Kaiser,et al.  Purification and characterization of a calcium oxalate monohydrate crystal growth inhibitor from human kidney tissue culture medium. , 1981, The Journal of biological chemistry.

[22]  Y. Aoki.  Crystallization and characterization of a new protease in mitochondria of bone marrow cells. , 1978, The Journal of biological chemistry.

[23]  W. Boyce.  Organic matrix of human urinary concretions. , 1968, The American journal of medicine.

[24]  E. Lyon,et al.  Mechanisms of genesis and growth of calculi. , 1968, The American journal of medicine.

[25]  Bodo E. Knudsen,et al.  Surface‐enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS): A new proteomic urinary test for patients with urolithiasis , 2004, Journal of clinical laboratory analysis.

[26]  R. Ryall,et al.  Calcium oxalate crystal matrix extract: the most potent macromolecular inhibitor of crystal growth and aggregation yet tested in undiluted human urine in vitro , 2004, Urological Research.

[27]  U. Meinhardt,et al.  Simultaneous measurements of calcium oxalate crystal nucleation and aggregation: impact of various modifiers , 2004, Urological Research.

[28]  N. Buchholz,et al.  Proteins in renal stones and urine of stone formers , 1998, Urological Research.