The ability of concentrated formic acid to formylate reactive amino acid residues is known from previous reports. In contrast, solvents containing a low concentration of formic acid are generally recognized as a safe environment for proteomic applications. The primary objective of this study was to explain the excessive formylation rate in tryptic peptides that did not come into contact with concentrated formic acid. We found out that the peptide formylation was associated with dissolving the peptides in a solvent containing mere 0.1% formic acid. Similar conclusions were drawn after analyzing publicly available proteomic data. We further demonstrated that these unwanted modifications can be averted via handling the samples at a low temperature or, obviously, via replacing formic acid in the solvent with trifluoroacetic acid. These simple countermeasures can contribute to a reduction in the part of the MS/MS spectra that remain unassigned to a peptide sequence.

Department of Analytical Chemistry Faculty of Pharmacy Charles University Heyrovského 1203 8 500 05 Hradec Králové Czech Republic

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Easy, Robust, and Repeatable Online Acid Cleavage of Proteins in Mobile Phase for Fast Quantitative LC-MS Bottom-Up Protein Analysis─Application for Ricin Detection