Recent developments in top down mass spectrometry have enabled closely related 
histone variants and their modified forms to be identified and quantitated with 
unprecedented precision, facilitating efforts to better understand how histones 
contribute to the epigenetic regulation of gene transcription and other nuclear 
processes. It is therefore crucial that intact MS profiles accurately reflect 
the levels of variants and modified forms present in a given cell type or cell 
state for the full benefit of such efforts to be realized. Here we show that 
partial oxidation of Met and Cys residues in histone samples prepared by 
conventional methods, together with oxidation that can accrue during storage or 
during chip-based automated nanoflow electrospray ionization, confounds MS 
analysis by altering the intact MS profile as well as hindering 
posttranslational modification localization after MS/MS. We also describe an 
optimized performic acid oxidation procedure that circumvents these problems 
without catalyzing additional oxidations or altering the levels of 
posttranslational modifications common in histones. MS and MS/MS of HeLa cell 
core histones confirmed that Met and Cys were the only residues oxidized and 
that complete oxidation restored true intact abundance ratios and significantly 
enhanced MS/MS data quality. This allowed for the unequivocal detection, at the 
intact molecule level, of novel combinatorially modified forms of H4 that would 
have been missed otherwise. Oxidation also enhanced the separation of human core 
histones by reverse phase chromatography and decreased the levels of 
salt-adducted forms observed in ESI-FTMS. This method represents a simple and 
easily automated means for enhancing the accuracy and sensitivity of top down 
analyses of combinatorially modified forms of histones that may also be of 
benefit for top down or bottom up analyses of other proteins.