Evaluation of histone sequence and modifications by electrospray ionization mass spectrometry and tandem mass spectrometry

Abstract

The histones, together with other specialized proteins and DNA, form the extraordinarily complex structure of chromatin. Electrospray ionization (ESI) permits the promotion of such protein species into the gas phase as intact, multiply charged molecular species. Mass spectrometry (MS), using a linear quadrupole mass filter, permits measurement of the relative molecular mass of these intact species with precision and accuracy. The latter are sufficient to evaluate variations in the primary structure of the histones and the type and extent of the natural and induced multiple covalent modifications. The locations of modifications are revealed by tandem mass spectrometry using tandem linear quadrupole or ion trap instruments on the intact species or the modified peptides derived by selective proteolysis. Experiments in applying this technique to histones from K562, a human-derived cell line, have demonstrated variations in the profile of modification through the normal cell cycle and in the presence of agents that inhibit enzymes responsible for reversal of the modification. The authors are currently testing the hypothesis that ESI-MS will permit the sensitive and selective identification of insult-induced modifications, distinguishing them from natural cell-cycle changes. This will be possible because ESI-MS reveals the full details of the profile of multiple posttranslational modificationsmore » of histones.« less