It is established that noncovalent complexes can be maintained both during and 
after electrospray and that assemblies of increasing size and complexity often 
lead to broadened peaks in mass spectra. This broadening arises from the 
tendency of large protein assemblies to form adducts with salts and is 
compounded when complexes are isolated directly from cells, without the full 
protein complement. To investigate the origins of this broadening in mass 
spectral peaks and to develop the optimal method for analyzing mass spectra of 
large protein complexes, we have carried out a systematic investigation of a 
series of noncovalent complexes representing a range of different sizes and 
architectures. We establish a positive correlation between peak width and the 
increased mass observed and show that this correlation is independent of the 
instrumental parameters employed. Using this relationship we show that we can 
determine masses of both 30S subunits and intact 2.3 MDa 70S ribosomes from 
Thermus thermophilus. The masses of both particles are consistent with multiple 
populations of ribosomes. To identify these various populations we combine 
simulated mass spectra of ribosomes, with and without the full protein 
complement, and estimate the extent of adducts from our study of known 
complexes. The results allow us to determine the contribution of the different 
subpopulations to the overall mass spectrum. We confirm the existence of these 
subpopulations using tandem mass spectrometry of intact 30S subunits. Overall, 
the results show that, rather than uniform particles, gas-phase ribosomes 
consist of a number of discrete populations. More generally, the results 
establish a rigorous procedure for accurate mass measurement and spectral 
analysis of heterogeneous macromolecular assemblies.