HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M600404-MCP200v1 6/9/1510    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pesavento, J. J. Articles by Mizzen, C. A. Search for Related Content PubMed PubMed Citation Articles by Pesavento, J. J. Articles by Mizzen, C. A. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 6:1510-1526, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

Mild Performic Acid Oxidation Enhances Chromatographic and Top Down Mass Spectrometric Analyses of Histones^*,S

James J. Pesavento^,, Benjamin A. Garcia^¶, James A. Streeky^¶, Neil L. Kelleher^,¶,|| and Craig A. Mizzen^||,**,

From the Center for Biophysics and Computational Biology, Departments of ^¶ Chemistry and ^** Cell and Developmental Biology, and ^|| Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				P. Rodriguez-Collazo, S. H. Leuba, and J. Zlatanova Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications Nucleic Acids Res., June 1, 2009; 37(11): e81 - e81. [Abstract] [Full Text] [PDF]

				A. C. Vendel, J. Calemine-Fenaux, A. Izrael-Tomasevic, V. Chauhan, D. Arnott, and D. L. Eaton B and T Lymphocyte Attenuator Regulates B Cell Receptor Signaling by Targeting Syk and BLNK J. Immunol., February 1, 2009; 182(3): 1509 - 1517. [Abstract] [Full Text] [PDF]

				J. J. Pesavento, C. R. Bullock, R. D. LeDuc, C. A. Mizzen, and N. L. Kelleher Combinatorial Modification of Human Histone H4 Quantitated by Two-dimensional Liquid Chromatography Coupled with Top Down Mass Spectrometry J. Biol. Chem., May 30, 2008; 283(22): 14927 - 14937. [Abstract] [Full Text] [PDF]

				H. Yang, J. J. Pesavento, T. W. Starnes, D. E. Cryderman, L. L. Wallrath, N. L. Kelleher, and C. A. Mizzen Preferential Dimethylation of Histone H4 Lysine 20 by Suv4-20 J. Biol. Chem., May 2, 2008; 283(18): 12085 - 12092. [Abstract] [Full Text] [PDF]

				J. J. Pesavento, H. Yang, N. L. Kelleher, and C. A. Mizzen Certain and Progressive Methylation of Histone H4 at Lysine 20 during the Cell Cycle Mol. Cell. Biol., January 1, 2008; 28(1): 468 - 486. [Abstract] [Full Text] [PDF]