Abstract
With the advent of high-resolution mass spectrometry together with sophisticated data analysis and interpretation algorithms, determination of protein synthesis and degradation rates (i.e. protein turnover) on a proteome-wide scale by employing stable isotope-labelled amino acids has become feasible. These dynamic data provide a deeper understanding of protein homeostasis and stress response mechanisms in microorganisms than well-established ‘steady state’ proteomics approaches. In this article, we summarize the technological challenges and solutions both on the biochemistry/mass spectrometry and bioinformatics level for turnover proteomics with a focus on chromatographic techniques. Although the number of available case studies for Corynebacterium glutamicum and related actinobacteria is still very limited, our review illustrates the potential of protein turnover studies for an improved understanding of questions in the area of biotechnology and biomedicine. Here, new insights from investigations of growth phase transition and different stress dynamics including iron, acid and heat stress for pathogenic but also for industrial actinobacteria are presented. Finally, we will comment on the advantages of integrated software solutions for biologists and briefly discuss the remaining technical challenges and upcoming possibilities for protein turnover analysis.
Highlights
Even before the advent of powerful proteome separation techniques like 2D-electrophoresis and the invention of biological mass spectrometry, the simple pulse-chase analysis of total cellular protein delivered a fundamental insight into bacterial physiology: a generally low protein degradation rate for growing bacteria experiencing ideal nutrient supply (Larrabee et al, 1980), and substantial reuse of amino acids through proteolysis under hunger conditions (Pine, 1972), which may reach up to 30% h-1 of total intracellular protein
The same group utilized 15N-arginine to monitor protein turnover in Mycobacterium tuberculosis. Another trick to obtain protein synthesis and degradation data was to use a combination of SILAC and isobaric tag for relative and absolute quantification (iTRAQ)-labelling [iTRAQ is a chemical label that allows relative quantification on the MS/MS-level (Aggarwal et al, 2006)]
Apart from its high price, the iTRAQ approach suffers from a limited dynamic range and peak overlaps on the MS and MS/MS level, which can lead to false quantification values. (Rao and colleagues, 2008a) published another strategy aiming at the parallel determination of synthesis and degradation based on MS chromatogram alignment
Summary
With the advent of high-resolution mass spectrometry together with sophisticated data analysis and interpretation algorithms, determination of protein synthesis and degradation rates (i.e. protein turnover) on a proteome-wide scale by employing stable isotopelabelled amino acids has become feasible. These dynamic data provide a deeper understanding of protein homeostasis and stress response mechanisms in microorganisms than well-established ‘steady state’ proteomics approaches. We summarize the technological challenges and solutions both on the biochemistry/mass spectrometry and bioinformatics level for turnover proteomics with a focus on chromatographic techniques.
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