Quantitative assessment of methyl-esterification and other side reactions in a standard propionylation protocol for detection of histone modifications

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• 作者：Veerle Paternoster ; Anders Valdemar Edhager ; Christian Sibbersen ; Anders Lade Nielsen ; Anders Dupont Bø ; rglum ; Jane Hvarregaard Christensen and Johan Palmfeldt
• 刊名：PROTEOMICS
• 出版年：2016
• 出版时间：July 2016
• 年：2016
• 卷：16
• 期：14
• 页码：2059-2063
• 全文大小：541K
• ISSN：1615-9861

文摘

Histone modifications play an important role in regulating chromatin stability and gene expression, but to date, investigating them remains challenging. In order to obtain peptides suitable for MS-based analysis, chemical derivatization of N-terminus and lysine residues by propionic anhydride is commonly performed. Several side reactions (methyl-esterification, amidation, solvolysis, overpropionylation, and missed propionylation) during propionylation protocols have been described, yet their relative abundances remain vague. Because methyl-esterification could interfere with correct interpretation of the modification pattern, it is essential to take measures to avoid it. Here we present in-depth quantitative analyses of methyl-esterification and the other side reactions in a standard propionylation protocol containing methanol, and when replacing methanol with isopropanol or acetonitrile. We show that the use of alternative solvents can eliminate methyl-esterification and that even though other side reactions are not prevented, their contribution can be kept relatively small. We also show that replacing methanol can be of importance also in other proteomics methods, such as mixed cation exchange, using methanol under acidic conditions.