Abstract

Liquid chromatography coupled to tandem mass spectrometry in combination with stable-isotope labeling is an established and widely spread method to measure gene expression on the protein level. However, it is often not considered that two opposing processes are responsible for the amount of a protein in a cell--the synthesis as well as the degradation. With this work, we provide an integrative, high-throughput method--from the experimental setup to the bioinformatics analysis--to measure synthesis and degradation rates of an organism's proteome. Applicability of the approach is demonstrated with an investigation of heat shock response, a well-understood regulatory mechanism in bacteria, on the biotechnologically relevant Corynebacterium glutamicum. Utilizing a multilabeling approach using both heavy stable nitrogen as well as carbon isotopes cells are metabolically labeled in a pulse-chase experiment to trace the labels' incorporation in newly synthesized proteins and its loss during protein degradation. Our work aims not only at the calculation of protein turnover rates but also at their statistical evaluation, including variance and hierarchical cluster analysis using the rich internet application QuPE.

Highlights

  • From the ‡Department of Plant Biochemistry, Ruhr-University Bochum, 44780 Bochum, Germany; §Computational Genomics, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany; ¶Biodata Mining Group, Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany; ʈBioinformatics Resource Facility, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany

  • Pratt et al [12] used stable isotope labeling by amino acids in cell culture (SILAC)1 [13] and matrix assisted laser desorption/time-of-flight (MALDI-TOF) mass spectrometry to determine degradation rates for approx. 50 proteins in glucose-limited yeast cells grown in an aerobic chemostat at steady state

  • Aiming at a proteome-wide applicable method to analyze the components of protein turnover—protein synthesis and degradation—we designed and implemented a pulse-chase experiment based on a multilabeling strategy that incorporates both 15N as well as 13C

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Summary

Introduction

From the ‡Department of Plant Biochemistry, Ruhr-University Bochum, 44780 Bochum, Germany; §Computational Genomics, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany; ¶Biodata Mining Group, Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany; ʈBioinformatics Resource Facility, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany. Degradation can be investigated by setting the protein amounts before and after an induced pulse into relation In this manner, Pratt et al [12] used stable isotope labeling by amino acids in cell culture (SILAC)1 [13] and matrix assisted laser desorption/time-of-flight (MALDI-TOF) mass spectrometry to determine degradation rates for approx. For organisms that have a comparably fast protein turnover, which is especially the case for bacteria, it is safe to assume that in all cases either a fully labeled or a fully unlabeled peptide is available This can be used for protein identification. We developed a new approach to gain these protein turnover ratios from isotopically labeled LC-MS/MS data in a highthroughput manner which is, first, well suited for fast-growing organisms such as bacteria and, second, does not impose any restrictions on sample handling and chromatographic setup. It was our aim to provide an integrated, user-friendly, and instantly accessible software solution, which allows the calculation of synthesis and degradation ratios and their in-depth statistical evaluation

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