Mild performic acid oxidation enhances chromatographic and top down mass spectrometric analyses of histones.
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.
Pesavento
James J
JJ
Center for Biophysics and Computational Biology, Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Garcia
Benjamin A
BA
Streeky
James A
JA
Kelleher
Neil L
NL
Mizzen
Craig A
CA
D002845:Chromatography / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005561:Formates / Q000737:chemistry*; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
D004220:Disulfides; D005561:Formates; D006657:Histones; D010455:Peptides; C008817:peroxyformic acid; D006861:Hydrogen Peroxide; D003545:Cysteine; D010100:Oxygen