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Quantification of N 6 ‐formylated lysine in bacterial protein digests using liquid chromatography/tandem mass spectrometry despite spontaneous formation and matrix effects
Author(s) -
Folz Jacob S.,
Patterson Jenelle A.,
Hanson Andrew D.,
Fiehn Oliver
Publication year - 2021
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.9019
Subject(s) - lysine , chemistry , chromatography , tandem mass spectrometry , mass spectrometry , acetylation , biochemistry , amino acid , gene
Rationale N 6 ‐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. Methods N 6 ‐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 N 6 ‐formyl lysine. Results We show that N 6 ‐formyl lysine, N 2 ‐formyl lysine, N 6 ‐acetyl lysine, and N 2 ‐acetyl lysine are all formed spontaneously during sample preparation and LC/MS/MS analysis, which complicates quantification of these metabolites in biological samples. N 6 ‐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 N 6 ‐formyl lysine, and 0.005% of lysine was converted into N 6 ‐formyl lysine in pure run solvent. Conclusions Spontaneous formation of N 6 ‐formyl lysine, N 6 ‐acetyl lysine, N 2 ‐formyl lysine, and N 2 ‐acetyl lysine needs to be subtracted from biologically formed lysine modifications when quantifying these epimetabolites in biological samples.