For more complete characterization of DNA-predicted proteins (including their 
posttranslational modifications) a "top-down" approach using high-resolution 
tandem MS is forwarded here by its application to methanogens in both 
hypothesis-driven and discovery modes, with the latter dependent on new 
automation benchmarks for intact proteins. With proteins isolated from ribosomes 
and whole-cell lysates of Methanococcus jannaschii (approximately 1,800 genes) 
using a 2D protein fractionation method, 72 gene products were identified and 
characterized with 100% sequence coverage via automated fragmentation of intact 
protein ions in a custom quadrupole/Fourier transform hybrid mass spectrometer. 
Three incorrect start sites and two modifications were found, with one of each 
determined for MJ0556, a 20-kDa protein with an unknown methylation at 
approximately 50% occupancy in stationary phase cells. The separation approach 
combined with the quadrupole/Fourier transform hybrid mass spectrometer allowed 
targeted and efficient comparison of histones from M. jannaschii, Methanosarcina 
acetivorans (largest Archaeal genome, 5.8 Mb), and yeast. This finding revealed 
a striking difference in the posttranslational regulation of DNA packaging in 
Eukarya vs. the Archaea. This study illustrates a significant evolutionary step 
for the MS tools available for characterization of WT proteins from complex 
proteomes without proteolysis.