title: Quantitative assessment of methyl-esterification and other side reactions in a standard propionylation protocol for detection of histone modifications.
abstract: 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.
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.|The Lundbeck Foundation Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus and Copenhagen, Denmark.|Centre for Integrative Sequencing, iSEQ, Aarhus University, Denmark.
authors.forename: Veerle
authors.initials: V
authors.lastname: Paternoster
authors.affiliation: Research Unit for Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Anders Valdemar
authors.initials: AV
authors.lastname: Edhager
authors.affiliation: Department of Chemistry, Aarhus University, Aarhus, Denmark.|Department of Forensic Medicine, Bioanalytical Unit, Aarhus University, Aarhus, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Sibbersen
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: Anders Lade
authors.initials: AL
authors.lastname: Nielsen
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.|The Lundbeck Foundation Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus and Copenhagen, Denmark.|Centre for Integrative Sequencing, iSEQ, Aarhus University, Denmark.
authors.forename: Anders Dupont
authors.initials: AD
authors.lastname: Børglum
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark.|The Lundbeck Foundation Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus and Copenhagen, Denmark.|Centre for Integrative Sequencing, iSEQ, Aarhus University, Denmark.
authors.forename: Jane Hvarregaard
authors.initials: JH
authors.lastname: Christensen
authors.affiliation: Research Unit for Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Johan
authors.initials: J
authors.lastname: Palmfeldt
mesh_terms: D019840:2-Propanol / Q000737:chemistry; D000097:Acetonitriles / Q000737:chemistry; D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000812:Anhydrides / Q000737:chemistry* / Q000378:metabolism; D016477:Artifacts; D004951:Esterification; D042421:Histone Code; D006657:Histones / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D000432:Methanol / Q000737:chemistry; D008745:Methylation; D010446:Peptide Fragments / Q000032:analysis*; D010449:Peptide Mapping; D011422:Propionates / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D012997:Solvents / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000592:standards; D014357:Trypsin / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D000577:Amides; D000596:Amino Acids; D000812:Anhydrides; D006657:Histones; D010446:Peptide Fragments; D011422:Propionates; D012997:Solvents; C096126:propionic anhydride; D014357:Trypsin; D019840:2-Propanol; D000432:Methanol; C032159:acetonitrile
keywords: Biomedicine; Histone; Mass spectrometry; Methanol; Posttranslational modifications; Propionylation