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Quantitative analysis of histone modifications: formaldehyde is a source of pathological n(6)-formyllysine that is refractory to histone deacetylases.

Edrissi B, Taghizadeh K, Dedon PC - PLoS Genet. (2013)

Bottom Line: While isotope labeling studies revealed that lysine demethylation is not a source of N(6)-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6)-formyllysine, with use of [(13)C,(2)H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources.Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6)-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct.These data suggest that N(6)-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

ABSTRACT
Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N(6)-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3'-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N(6)-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N(6)-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N(6)-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1-4 modifications per 10(4) lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10(4) lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N(6)-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6)-formyllysine, with use of [(13)C,(2)H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6)-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct. These data suggest that N(6)-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification.

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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.
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pgen-1003328-g002: 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.

Mentions: Our previous method for quantifying N6-formyllysine in proteins involved proteinase K-mediated hydrolysis of proteins, derivatization of the resulting amino acids with phenylisothiocyanate (PITC), HPLC pre-purification of amino acid derivatives, and final LC-MS/MS analysis of the derivatized amino acids [9]. This method proved to be relatively insensitive and biased as a result of using proteinase K, which produced only partial hydrolysis of some proteins when used in small quantities to minimize background autolysis. To resolve these problems, we used Streptomyces griseus protease at ratio of 1 µg enzyme per 10 µg proteins, which resulted in efficient and complete digestion of all proteins as judged by comparing the measured amount of lysine released per µg of purified histone proteins to the theoretical lysine content of the proteins. In addition, the method was optimized to eliminate HPLC pre-purification step, and the need for PITC derivatization to achieve chromatographic resolution of amino acids was obviated by use of aqueous normal phase HPLC with a diamond-hydride column. This chromatographic system resolved N6-acetyllysine, mono-, di-, and tri-N6-methyllysines, as well as N6-formyllysine and lysine, as shown in Figure 2. With isotopically labeled internal standards added prior to protease digestion, identification and quantification of amino acids were accomplished by HPLC-coupled to tandem quadrupole mass spectrometry in positive ion mode, using multiple reaction monitoring (MRM) transitions. With a 2% precision for technical replicates, the limits of detection were found to be 1 fmol for N6-formyl- and N6-acetyllysine, 10 fmol for lysine, and 50 fmol for each of N6-mono-, di-, and tri-methyl lysine. Data for the various lysine modifications are expressed here as proportions of the total number of lysines in the sample.


Quantitative analysis of histone modifications: formaldehyde is a source of pathological n(6)-formyllysine that is refractory to histone deacetylases.

Edrissi B, Taghizadeh K, Dedon PC - PLoS Genet. (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585032&req=5

pgen-1003328-g002: 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.
Mentions: Our previous method for quantifying N6-formyllysine in proteins involved proteinase K-mediated hydrolysis of proteins, derivatization of the resulting amino acids with phenylisothiocyanate (PITC), HPLC pre-purification of amino acid derivatives, and final LC-MS/MS analysis of the derivatized amino acids [9]. This method proved to be relatively insensitive and biased as a result of using proteinase K, which produced only partial hydrolysis of some proteins when used in small quantities to minimize background autolysis. To resolve these problems, we used Streptomyces griseus protease at ratio of 1 µg enzyme per 10 µg proteins, which resulted in efficient and complete digestion of all proteins as judged by comparing the measured amount of lysine released per µg of purified histone proteins to the theoretical lysine content of the proteins. In addition, the method was optimized to eliminate HPLC pre-purification step, and the need for PITC derivatization to achieve chromatographic resolution of amino acids was obviated by use of aqueous normal phase HPLC with a diamond-hydride column. This chromatographic system resolved N6-acetyllysine, mono-, di-, and tri-N6-methyllysines, as well as N6-formyllysine and lysine, as shown in Figure 2. With isotopically labeled internal standards added prior to protease digestion, identification and quantification of amino acids were accomplished by HPLC-coupled to tandem quadrupole mass spectrometry in positive ion mode, using multiple reaction monitoring (MRM) transitions. With a 2% precision for technical replicates, the limits of detection were found to be 1 fmol for N6-formyl- and N6-acetyllysine, 10 fmol for lysine, and 50 fmol for each of N6-mono-, di-, and tri-methyl lysine. Data for the various lysine modifications are expressed here as proportions of the total number of lysines in the sample.

Bottom Line: While isotope labeling studies revealed that lysine demethylation is not a source of N(6)-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6)-formyllysine, with use of [(13)C,(2)H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources.Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6)-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct.These data suggest that N(6)-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

ABSTRACT
Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N(6)-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3'-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N(6)-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N(6)-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N(6)-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1-4 modifications per 10(4) lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10(4) lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N(6)-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6)-formyllysine, with use of [(13)C,(2)H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6)-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct. These data suggest that N(6)-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification.

Show MeSH
Related in: MedlinePlus