Eukaryotic histones serve as prototypical examples of posttranslational 
complexity with diverse modifications (PTMs) on many different residues that 
comprise a "Histone Code". To help crack this code more efficiently, we 
demonstrate a new strategy for protein characterization wherein complete PTM 
descriptions are obtained by database retrieval instead of manual interpretation 
of information-rich data from high-resolution tandem mass spectrometry (MS/MS). 
A database of nearly 50 000 modified histone H4 sequences was created and 
queried with 91 fragment ions from electron capture dissociation of a histone 
form +112 Da (versus unmodified mass) selectively accumulated in a quadrupole 
Fourier transform hybrid mass spectrometer. The correct form atop the retrieval 
list indicated dimethylation at Lys20, acetylation at the N terminus, and 
acetylation at Lys16 (resolved from trimethylation, Deltam = 0.036 Da). A 
statistical evaluation reveals the critical role of mass accuracy and that PTM 
"isomers" are retrieved as next-best matches. The applicability of shotgun 
annotation to forms of H4 with up to six PTMs is demonstrated, with 
extensibility to other histones (e.g., H2A, H2B, H3) and other protein classes 
projected.