Fig. 1
Histone homocysteinylation is a common modification among different tissues and species. a A typical HPLC-MS/MS spectra of a tryptic peptide ‘RGGVKHcyRISGLIYEETR’ harboring H4K44 homocystylation, derived from human brain. The x and y axes represent m/z and relative ion intensity, respectively. A series of b- and y-type homocysteinylation fragment ions are evident which not only provide reliable sequence information, but also indicate an unambiguous +174.04600 Da shift for Hcy. b Schematic illustration of homocysteinylation sites of histone lysine residues in human normal brain samples identified using HPLC-MS/MS. The red diamond shape depicts homocysteinylation sites in core histones (H3, H4, H2a, and H2b). The number underneath each red lysine residue (K) represents the position of the particular lysine residue within each respective histone. c Verification of anti-KHcy antibody. The homocysteinylation levels of BSA (Bovine Serum Album) and KHcy modified BSA were detected with anti-KHcy antibody under the presence of 0, 2, or 5 µg/ml of Hcy modified lysine (KHcy as competitor). CBB Coomassie Brilliant Blue staining. These test repeated for 3 times and the quantitation of the western blotting showed on right. In the BSA group, the relative K-Hcy levels were 1 ± 0.05, 1.15 ± 0.10; 1.11 ± 0.78. In the BSA-Hcy group, the relative K-Hcy levels were 10.88 ± 1.02, 5.48 ± 0.34; 1.39 ± 0.21. d Verification of specificity of the anti-K-Hcy antibody. Western blotting assay was carried out by incubating the anti-KHcy antibody with unmodified OVA (ovalbumin), acetylated-OVA, succinylated-OVA, or K-Hcy-OVA. e Western blotting analysis for the detection of H3 homocystylations in samples from a variety of human fetal tissues, including brain, spinal cord, heart, liver, lung, kidney, muscle, and skin. Anti-Hcy: rabbit polyclonal anti-Hcy antibody; Anti-H3: rabbit polyclonal anti-H3 antibody. f Presence of H3 homocysteinylation in different species, including D. melanogaster, Zebra fish, Gallus gallus brain, mouse brain, and human fetal brain, demonstrated using western blotting with rabbit polyclonal anti-Hcy and anti-H3 antibodies
