Detailed characterization of the amino acid (aa) compositions of recombinant 
histone H3 (Xenopus laevis) and its Lys-9 mono-, di-, and trimethylated isoforms 
was carried out by ESI/FT-ICR-MS. The measured molecular masses of these four 
proteins did not agree with those predicted from the published wild-type histone 
H3 sequence. Using MS/MS with both collision-activated dissociation and electron 
capture dissociation, three aa substitutions (Gly102Ala, Cys110Ala, and 
Gly111Ala) were unambiguously identified in each protein by protein database 
searching and de novo peptide tag sequencing. In addition, it was determined 
through accurate mass measurement and elemental composition generation that 
Lys-9, to which the methyl groups are attached, is not in fact a lysine. 
Instead, it was identified as a chemical analog of 
lysine--aminoethylcysteine--in the methylated proteins. After incorporation of 
these three aa substitutions and the aminoethylcysteine into the protein 
sequences, the re-calculated molecular masses of four proteins were completely 
consistent with the measured values within 1 ppm. This work demonstrates the 
power of top-down MS for a detailed structural confirmation of recombinant 
proteins, even without prior information on aa substitutions or modifications.