Figure 1.
The adduct can be generated in chromatin proteins from reaction of lysine with 3′-formylphosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with endogenous or exogenous formaldehyde. Formaldehyde reacts with amines to give a carbinolamine intermediate (N6-(hydroxymethyl)-lysine) that is in equilibrium with a Schiff base and that is one oxidation state away from the formamide functional group of N6-formyllysine.
Figure 2.
Different lysine species detected in purified histone H4 from TK6 cells.
Lysine adducts were monitored by tandem mass spectrometry, as described in Materials and Methods. Abbreviations: FK, N6-formyllysine; AK, N6-acetyllysine; K, lysine; MK, N6-mono-methyllysine; M2K, N6-di-methyllysine; M3K, N6-tri-methyllysine.
Table 1.
Quantification of lysine modifications in HPLC-purified histone proteins.1
Table 2.
Quantification of N6-formyllysine in different proteins.
Figure 3.
Formaldehyde is a source of N6-formyllysine.
Formation of N6-formyllysine in (A) in vitro reactions of 1 mM L-lysine with formaldehyde for 2 h at 37°C, and in (B) TK6 cells exposed to formaldehyde, as described in Materials and Methods. Data represent mean ± SD for N = 3, with asterisks denoting statistically significant differences by Student's t-test (p<0.05).
Figure 4.
Addition of [13C,2H2]-formaldehyde to TK6 cells distinguishes exogenous from endogenous sources of N6-formyllysine.
(A) LC-MS/MS analysis showing signals for the three isotopomeric N6-formyllysine species, as described in Materials and Methods. (B) Plot of N6-formyllysine levels as a function of exposure to [13C,2H2]-formaldehyde. Data represent mean ± SD for N = 3.
Figure 5.
Analysis of lysine demethylation as a source of N6-formyllysine.
Methyl groups in N6-methyllysine species in TK6 cells were labeled using L-methionine-([13C,2H3]-methyl) and N6-formyllysine and N6-methyllysine species were quantified by LC-MS/MS as described in Materials and Methods. Panels A and B: N6-mono-methyllysine and N6-di-methyllysine are predominately labeled (>90%) with heavy isotope methyl groups (mass increase of 4 m/z and 8 m/z, respectively), with <10% of the modifications containing unlabeled methyl groups. Panel C: the level of N6-[13C, 2H]-formyllysine (177 m/z→114 m/z transition) in histones did not show an increase beyond the natural isotope abundance level of ∼0.7% for [M+2] ion of N6-formyllysine.
Figure 6.
Effect of lysine deacetylases on N6-formyllysine.
(A) TK6 cells were treated with the class I and class II histone deacetylase inhibitor, SAHA, as described in Materials and Methods. Data represent mean ± SD for N = 3, with asterisks denoting statistically significant differences by Student's t-test (p<0.05). (B) Treatment of peptide substrates containing N6-acetyllysine or N6-formyllysine with the class III histone deacetylase, SIRT1.
Figure 7.
Summary of findings on N6-formyllysine in histones.
N6-formyllysine can arise from reaction of lysine with the 3′-formyl phosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with formaldehyde. Furthermore, our data suggest that N6-formyllysine is refractory to removal by histone deacetylases, which is consistent with the persistence of this pathological adduct throughout the life of individual histone proteins.
Table 3.
Summary of mass spectrometry parameters for each species.