title: Characterizations of heparin-binding proteins in human urine by affinity purification-mass spectrometry and defining "L-x(2,3)-A-x(0,1)-L" as a novel heparin-binding motif.
abstract: Heparin-binding proteins (HBPs) are considered as potential modulators of kidney stone formation. However, HBPs had not been characterized in the urine previously. In this study, we applied affinity purification-mass spectrometry (AP-MS) using cellufine sulfate column chromatography and liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-Q-TOF MS/MS) to identify HBPs in normal human urine. Using this approach, 83 HBPs were identified, including those involved in metabolic process, cellular process, immune system, developmental process, response to stimuli, cell communication, transport, cell adhesion and others. The AP-MS data were confirmed by Western blot analysis and chemico-protein interactions analysis using STITCH tool. In addition, 59, 55 and 51 identified HBPs had the known heparin-binding motifs "XBBXnBX", "XBXnBBX" and "XBBBnX", respectively. Moreover, a novel heparin-binding motif "L-x(2,3)-A-x(0,1)-L" was found in 58 identified HBPs using PRATT tool. The sensitivity and specificity of this novel motif were 85% and 100%, respectively, by validation using 20 known HBPs and 11 non-HBPs. We report herein for the first time a large number of HBPs in normal human urine and defined "L-x(2,3)-A-x(0,1)-L" as a novel heparin-binding motif. These findings will be useful to further understand the renal physiology and may also lead to identification of novel modulators of kidney stone formation.
  
  
  
  Heparin-binding proteins (HBPs) have several important roles in various biological processes, including kidney stone formation. However, HBPs had not been characterized in the urine. Our present work using affinity purification coupled to mass spectrometry (AP-MS) is the first large-scale study on HBPs in human urine. In addition to the three known heparin-binding motifs, "XBBXnBX", "XBXnBBX", and "XBBBnX", we successfully defined the amino acid pattern "L-x(2,3)-A-x(0,1)-L" as a novel heparin-binding motif. These findings will be useful to further understand the renal physiology and may also lead to identification of novel modulators of kidney stone formation.
authors.affiliation: Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital; and Center for Research in Complex Systems Science, Mahidol University, Bangkok, Thailand.
authors.forename: Juthatip
authors.initials: J
authors.lastname: Manissorn
authors.affiliation: Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital; and Center for Research in Complex Systems Science, Mahidol University, Bangkok, Thailand. Electronic address: thongboonkerd@dr.com.
authors.forename: Visith
authors.initials: V
authors.lastname: Thongboonkerd
mesh_terms: D002846:Chromatography, Affinity; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D014556:Urine / Q000737:chemistry
chemical_list: D011506:Proteins; D006493:Heparin
keywords: AP-MS; Cellufine sulfate; Heparin-binding; Kidney stone; Motif; Proteome; Proteomics; Urine