title: Preventing N- and O-formylation of proteins when incubated in concentrated formic acid.
abstract: Concentrated formic acid is among the most effective solvents for protein solubilization. Unfortunately, this acid also presents a risk of inducing chemical modifications thereby limiting its use in proteomics. Previous reports have supported the esterification of serine and threonine residues (O-formylation) for peptides incubated in formic acid. However as shown here, exposure of histone H4 to 80% formic (1 h, 20(o) C) induces N-formylation of two independent lysine residues. Furthermore, incubating a mixture of Escherichia coli proteins in formic acid demonstrates a clear preference toward lysine modification over reactions at serine/threonine. N-formylation accounts for 84% of the 225 uniquely identified formylation sites. To prevent formylation, we provide a detailed investigation of reaction conditions (temperature, time, acid concentration) that define the parameters permitting the use of concentrated formic acid in a proteomics workflow for MS characterization. Proteins can be maintained in 80% formic acid for extended periods (24 h) without inducing modification, so long as the temperature is maintained at or below -20(o) C.
authors: Department of Chemistry, Dalhousie University, Halifax, NS, Canada.|Key Laboratory of Pesticides and Chemical Biology, College of Chemistry, Central China Normal University, Wuhan, Hubei, P. R. China.
authors: Shi
authors: S
authors: Zheng
authors: Department of Chemistry, Dalhousie University, Halifax, NS, Canada.
authors: Alan A
authors: AA
authors: Doucette
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D005561:Formates / Q000737:chemistry*; D006657:Histones; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012694:Serine / Q000737:chemistry*; D012995:Solubility; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D005561:Formates; D006657:Histones; D010446:Peptide Fragments; C030544:formic acid; D013912:Threonine; D012694:Serine
keywords: Formic acid; N-formylation; O-formylation; Protein modifications; Protein solubilization; Technology