Comprehensive Characterization of Posttranslational Modifications of Histones Using a Novel High-Throughput Middle-Down Proteomics Approach

Abstract

Until recently, histones have mostly been characterized by either bottom-up or top-down mass spectrometric analysis. While the bottom-up approach is more sensitive, the important connectivity of the posttranslational modifications of the N-terminus is often lost. This connectivity is maintained in top-down proteomics, but the sensitivity of this approach is lacking. Middle-down proteomics is a compromise between bottom-up and top-down analysis using a limited digestion that creates peptides large enough to maintain the important connectivity in the N-terminal tail of histones. To achieve a high-throughput method, middle-down proteomics was coupled with on-line chromatographic separation using electron capture dissociation (ECD), a fragmentation technique that preserves the posttranslational modifications. Taking advantage of the high mass accuracy in the FTICR mass analyzer, several histone variants were identified and modification sites were successfully localized, including single, multiple and positional isomeric PTM sites. In general, protein sequence coverage was well over 50%, reaching 100% in many instances.