Top-down analysis of recombinant histone H3 and its methylated analogs by ESI/FT-ICR mass spectrometry.
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.
Han
Jun
J
University of Victoria-Genome BC Proteomics Centre, University of Victoria, Victoria, BC, Canada.
Borchers
Christoph H
CH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D006657:Histones / Q000032:analysis* / Q000235:genetics; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000295:instrumentation / Q000379:methods; D014982:Xenopus laevis
D006657:Histones; D011994:Recombinant Proteins; D008239:Lysine