Dataset Information

Cellulose Acetate Membrane Electrophoresis Based Urinary Proteomics for the Identification of Characteristic Proteins.

ABSTRACT: BACKGROUND:Analysis of urinary proteins using cellulose acetate membrane electrophoresis (CAME) is a useful and challenging method for the recognition of damaged sites in the kidney. However, protein content of each CAME fraction is still not completely understood. METHODS:In this study, an effective method of protein extraction from each band fractionated by CAME was established, which enabled us to examine the extracted proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. RESULTS:Proteins were extracted from the gel and analyzed by mass spectrometry. In all, 31 proteins were identified, including 20 urinary proteins that were newly identified in the CAME-based analysis. CONCLUSION:This methodology was useful for identifying the proteins responsible for creating unique bands on CAME in a urine sample of a patient with drug-induced interstitial nephritis. These findings provide in-depth characterization of urinary protein contents in each CAME fraction.

SUBMITTER: Nakayama A

PROVIDER: S-EPMC6807239 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Cellulose Acetate Membrane Electrophoresis Based Urinary Proteomics for the Identification of Characteristic Proteins.

Nakayama Aki A Kubota Ryo R Sakatsume Minoru M Suzuki Hidenori H Katayama Akira A Kanamori Kiyoko K Shiba Kiyoko K Iijima Shiro S

Journal of clinical laboratory analysis 20151010 5

<h4>Background</h4>Analysis of urinary proteins using cellulose acetate membrane electrophoresis (CAME) is a useful and challenging method for the recognition of damaged sites in the kidney. However, protein content of each CAME fraction is still not completely understood.<h4>Methods</h4>In this study, an effective method of protein extraction from each band fractionated by CAME was established, which enabled us to examine the extracted proteins by sodium dodecyl sulfate-polyacrylamide gel elect ...[more]

PMID: 26453827

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Project description:The proteins of microvilli prepared from pig kidney were analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The typical pattern stained for protein revealed five major bands, four of which also stained for carbohydrate, and about 15 minor bands. For descriptive purposes the bands were designated numerically by their apparent molecular weights (X10(-3). Well-characterized proteins were identified with four of the five major bands. Dipeptidyl peptidase IV, a serine proteinase that may be specifically labelled with di-isopropyl [32P]phosphorofluoridate, was assigned to band 130. Aminopeptidase M was assigned to band 160, though when released from the membrane by a proteinase, this protein comprises three polypeptides each of lower apparent molecular weight than the native enzyme. Neutral endopeptidase can be assigned to band 95 and actin to band 42. The fifth major band (180) is an extrinsic glycoprotein that has not been identified with any microvillus enzyme activity. These four proteins contribute 21% of the microvillus-membrane protein. Kidney microvillus actin was characterized by a variety of properties and was similar to muscle actin. A computer analysis of the gel pattern indicates that it comprises 9.0% of the microvillus protein. Myosin is not present in the microvillus, but another protein associated with band 95, with properties that distinguish it from neutral endopeptidase, was tentatively identified as alpha-actinin. Alkaline phosphatase was identified as a monomeric polypeptide with an apparent molecular weight of 80000; it is a minor protein of the microvillus and is not discernible as a discrete band in the gel pattern. These and other results permit a model of the organization of the microvillus protein to be suggested. The computer program used has been deposited as Supplementary Publication SUP 50070 (12 pages) at the British Library Lending Division, Boston Spa. Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153,5.

Dataset Information

Cellulose Acetate Membrane Electrophoresis Based Urinary Proteomics for the Identification of Characteristic Proteins.

Publications

Cellulose Acetate Membrane Electrophoresis Based Urinary Proteomics for the Identification of Characteristic Proteins.

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