Top-down analysis of recombinant histone H3 and its methylated analogs by ESI/FTICR mass spectrometry

Detailed characterization of the amino acid compositions of recombinant histone H3 (Xenopus laevis) and its Lys-9 mono-, di- and tri-methylated isoforms was carried out by electrospray ionization/Fourier transform ion cyclotron resonance mass spectrometry (ESI/FTICR-MS). The measured molecular masses of these four proteins did not agree with those predicted from the published wild-type histone H3 sequence. Using tandem mass spectrometry with both collision-activated dissociation (CAD) and electron capture dissociation (ECD), three amino acid substitutions (Gly102Ala, Cys110Ala, Gly111la) 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 amino acid 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 demonstrated the power of top-down FTMS for detailed structural confirmation of recombinant proteins, even without prior information on amino acid substitutions or modifications.