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      Proteomic Identification of a Large Complement of Rat Urinary Proteins


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          The characterization of urinary proteins is an important tool to identify disease-related biomarkers and to better understand renal physiology. Expression of urinary proteins has been previously studied by Western blotting and other immunological methods. The scope of such studies, however, is limited to previously identified proteins for which specific antibodies are existed. We used proteomic analysis to identify proteins and to construct a proteome map for Sprague-Dawley (SD) rat urine isolated by ultracentrifugation. Urinary proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and visualized by silver staining. Proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), followed by peptide mass fingerprinting using the NCBI protein database. A total of 350 protein spots were visualized. From 250 excised spots, 111 protein components were identified including transporters, transport regulators, chaperones, enzymes, signaling proteins, cytoskeletal proteins, pheromone-binding proteins, receptors, and novel gene products. The presence of a number of these identified proteins was unexpected and had not previously been identified in the urine. 2-D Western blot analyses for randomly selected proteins (ezrin, HSP70, β- and γ-actin, Rho-GDI, and l-myc) clearly confirmed the proteomic identification. Several potential posttranslational modifications were predicted by bioinformatic analyses. These data indicate that a large complement of expected and unexpected urinary proteins can be simultaneously studied by proteomic analysis. This approach may lead to better understanding of renal physiology and pathophysiology, and to biomarker discovery.

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          Most cited references15

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          Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, α-enolase and heat shock cognate 71: Proteomics of oxidized proteins in AD brain

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            Proteomic identification of oxidatively modified proteins in alzheimer’s disease brain. part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1

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              Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion.

              Ezrin, a membrane-cytoskeleton linker, is required for cell morphogenesis, motility, and survival through molecular mechanisms that remain to be elucidated. Using the N-terminal domain of ezrin as a bait, we found that p125 focal adhesion kinase (FAK) interacts with ezrin. We show that the two proteins coimmunoprecipitate from cultured cell lysates. However, FAK does not interact with full-length ezrin in vitro, indicating that the FAK binding site on ezrin is cryptic. Mapping experiments showed that the entire N-terminal domain of FAK (amino acids 1-376) is required for optimal ezrin binding. While investigating the role of the ezrin-FAK interaction, we observed that, in suspended kidney-derived epithelial LLC-PK1 cells, overproduction of ezrin promoted phosphorylation of FAK Tyr-397, the major autophosphorylation site, creating a docking site for FAK signaling partners. Treatment of the cells with a Src family kinase inhibitor reduced the phosphorylation of Tyr-577 but not that of Tyr-397, indicating that ezrin-mediated FAK activation does not require the activity of Src kinases. Altogether, these observations indicate that ezrin is able to trigger FAK activation in signaling events that are not elicited by cell-matrix adhesion.

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                October 2003
                17 November 2004
                : 95
                : 2
                : e69-e78
                aCore Proteomics Laboratory, Kidney Disease Program, Department of Medicine, bDepartment of Biochemistry and Molecular Biology, University of Louisville, cVeterans Administration, Louisville, Ky., and dDepartment of Medicine, The Medical University of South Carolina, and eRalph H. Johnson VA Medical Center, Charleston, S.C., USA
                73674 Nephron Exp Nephrol 2003;95:e69–e78
                © 2003 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                : 02 December 2002
                : 28 July 2003
                Page count
                Figures: 3, Tables: 3, References: 28, Pages: 1
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/73674
                Self URI (text/html): https://www.karger.com/Article/FullText/73674
                Self URI (journal page): https://www.karger.com/SubjectArea/Nephrology
                Original Paper

                Cardiovascular Medicine,Nephrology
                Proteome,Proteomics,GAP-43,Kidney,<italic>L</italic>-myc,2-D Western blot,Posttranslational modifications,Ezrin,2-D PAGE,Biomarker


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