Preventing N- and O-formylation of proteins when incubated in concentrated formic acid.

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.