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. Isolated subunits were analyzed using limited proteolysis and MALDI-MS and the results were compared with previous data obtained from 70S ribosomes. Generally, ribosomal proteins were found to be more stable in 70S ribosomes than in their isolated subunits, consistent with a reduction in conformational flexibility on subunit assembly. This approach demonstrates that limited proteolysis combined with MALDI-MS can reveal structural changes to ribosomes on subunit assembly or disassembly, and provides the appropriate benchmark data from 30S, 50S, and 70S proteins to enable studies of ribosome assembly intermediates. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 410-422, 2009.