Shotgun annotation of histone modifications: a new approach for streamlined characterization of proteins by top down mass spectrometry

J Am Chem Soc. 2004 Mar 24;126(11):3386-7. doi: 10.1021/ja039748i.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Databases, Protein
  • Histones / chemistry*
  • Humans
  • Molecular Sequence Data
  • Protein Isoforms
  • Proteomics / methods*
  • Spectrometry, Mass, Electrospray Ionization / methods*

Substances

  • Histones
  • Protein Isoforms