Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research
Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.
Key wordsMass spectrometry Ribosome heterogeneity Stress Proteomics
We thank Birgit Klinkert and Johanna Roßmanith for the practical introduction into ribosome isolation and for technical support. Jennifer Stepanek and Dominik Wüllner are acknowledged for critically reading the manuscript. Furthermore, we would like to thank Dörte Becher and Knut Büttner for sharing mass spectrometry protocols. Funding from the German Federal State of North Rhine Westphalia (NRW) is acknowledged for the mass spectrometer (“Forschungsgroßgeräte der Länder”) used in this protocol. JEB acknowledges funding from NRW for the grant “Translation of innovative antibiotics from NRW.”
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