Analysis of Methylation, Acetylation, and Other Modifications in Bacterial Ribosomal Proteins

A wide variety of posttranslational modifications of expressed proteins are known to occur in living organisms (Krishna R, Wold F. Post-translational modification of proteins. In: Meister A (ed) Advances in enzymology and related areas of molecular biology. Wiley, New York, 1993, pp 265-296). Although their presence in an organism cannot be predicted from the genome, these modifications can play critical roles in protein structure and function. The identification of posttranslational modifications is critical to our understanding of the functions of proteins involved in important biological pathways and mass spectrometry offers a fast, accurate method for observing them. A combined top-down/bottom-up approach can be used for identification and localization of posttranslational modifications of ribosomal proteins. This chapter describes procedures for analyzing Escherichia coli ribosomal proteins and their modifications by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. It also covers the analysis of gram-negative Caulobacter crescentus and gram-positive Bacillus subtilis ribosomal proteins by electrospray quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Confirmation of the occurrence and localization of PTMs is obtained through mass spectrometric analysis of tryptic peptides.