Quantification of N⁶-formylated lysine in bacterial protein digests using liquid chromatography/tandem mass...

N⁶-Formyl lysine is a well-known modification of histones and other proteins. It can also be formed as a damaged product from direct formylation of free lysine and accompanied by other lysine derivatives such as acetylated or methylated forms. In relation to the activity of cellular repair enzymes in protein turnover and to lysine metabolism, it is important to accurately quantify the overall ratio of modified lysine to free lysine.

N⁶-Formyl lysine was quantified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with data collected in a non-targeted manner using positive mode electrospray ionization on a Q-Exactive HF⁺ Orbitrap mass spectrometer. Studies were performed with lysine and deuterated lysine spiked into protein digests and solvents to investigate the extent of spontaneous formation and matrix effects of formation of N⁶-formyl lysine.

We show that N⁶-formyl lysine, N²-formyl lysine, N⁶-acetyl lysine, and N²-acetyl lysine are all formed spontaneously during sample preparation and LC/MS/MS analysis, which complicates quantification of these metabolites in biological samples. N⁶-Formyl lysine was spontaneously formed and correlated to the concentration of lysine. In the sample matrix of protein digests, 0.03% of lysine was spontaneously converted into N⁶-formyl lysine, and 0.005% of lysine was converted into N⁶-formyl lysine in pure run solvent.

Spontaneous formation of N⁶-formyl lysine, N⁶-acetyl lysine, N²-formyl lysine, and N²-acetyl lysine needs to be subtracted from biologically formed lysine modifications when quantifying these epimetabolites in biological samples.