In vivo protein isoform turnover analysis using 15N metabolically-labeled mouse tissue

Sharon Liu, Christiane Rewerts, Giuseppina Maccarrone, Christoph Turck

Abstract


Protein turnover, the dynamic process of protein synthesis and degradation, is known to be modulated by structural variations in proteins arising from alternative splicing or post-translational modifications (PTMs). The present study aimed to identify protein isoforms of 15N metabolically-labeled mouse lung and heart tissue, and to estimate protein turnover with the novel, in-house software Proturnyzer. We extracted and homogenized heart and lung tissue from 3 different mice that had been partially metabolically-labeled with 15N. Protein isoforms were separated using 2D-gel electrophoresis, subjected to in-gel enzymatic digest followed by mass spectrometry-based identification. We were able to identify 17 and 19 distinct proteins with isoforms in the mouse heart and lung, respectively. PTMs were identified in some isoforms and differences in turnover among isoforms could be determined from tryptic peptide 15N incorporation rates. Collectively, this platform offers a potential framework for future work using protein turnover data as a novel biomarker to analyze subtle differences between healthy and diseased tissues.

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Copyright (c) 2014 Sharon Liu