Limited proteolysis analysis of the ribosome is affected by subunit association.

Our understanding of the structural organization of ribosome assembly intermediates, in particular those intermediates that result from misfolding leading to their eventual degradation within the cell, is limited because of the lack of methods available to characterize assembly intermediate structures. Because conventional structural approaches, such as NMR, X-ray crystallography, and cryo-EM, are not ideally suited to characterize the structural organization of these flexible and sometimes heterogeneous assembly intermediates, we have set out to develop an approach combining limited proteolysis with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) that might be applicable to ribonucleoprotein complexes as large as the ribosome. This study focuses on the limited proteolysis behavior of appropriately assembled ribosome subunits.

Effects of streptomycin resistance mutations on posttranslational modification of ribosomal protein S12.

Ribosomal protein S12 contains a highly conserved aspartic acid residue that is posttranslationally beta-methylthiolated. Using mass spectrometry, we have determined the modification states of several S12 mutants of Thermus thermophilus and conclude that beta-methylthiolation is not a determinant of the streptomycin phenotype.

Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry.

A protocol has been developed that allows protein identifications using available DNA-based or protein sequences from a reference strain of a bacterial species to be extended to bacterial strains for which no prior DNA-based or protein sequence information exists. The protocol is predicated on careful isolation of a specific sub-cellular group of proteins. In this study, ribosomal proteins were chosen due to their high relative abundance and similarity in copy number per cell.