(a) Specificity of the anti-H3T22ac antibody was demonstrated by dot blot assay using four synthetic peptides: H3T22, un-acetylated peptide KQLATKAAR; H3T22ac, acetylated peptide KQLATacKAAR; M8S253ac, acetylated peptide KSacPLTEPNFENKC; M8S71ac, acetylated peptide TSacITPSSQDICRICHCEGDC. undefined residues additional to lysine. infection, the deadly disease called bubonic plague, the serine and threonine residues of human proteins are indeed acetylated by the effector protein YopJ [20,21]. The bacterial YopJ protein acts as an acetyltransferase that binds and acetylates serine and threonine residues in the activation loops of several kinases, including mitogen-activated protein kinase kinases (MAPKKs) and IB kinase (IKK). Thus, YopJ inhibits their phosphorylation and consequently their activation [20,21]. Mass spectrometry (MS)-based proteomics has led to the discovery of a large number of new histone and non-histone post-translational modifications (PTMs), which can be detected by PTM-related diagnostic mass shifts of fragment ions in MS/MS spectra. Either histone H3 K56 acetylation (globular domain) at the entryCexit gate enabled recruitment of the SWI/SNF (SWItch/Sucrose Non-Fermentable) nucleosome remodeling complex and so regulated gene activity . In view of those findings, the putative new sites of acetylation which were mapped to the globular core domain might have important biological functions for gene expression. Open in a separate window Figure 3 Human histone acetylation sites identified by the wide tolerance acetylation workflow. A diagram showing sites of histone acetylation identified by the wide tolerance acetylation workflow. Red, acetylation sites; underlined, new in human histones; dotted box, the globular core domain; subscript, position of the amino acid residues; *, liver INHBB histone H1e. 3.4. Validation of T-Acetylation in the N-Terminal Tail of Human Histone H3 (H3T22ac) by Immune Assays To validate the wide tolerance acetylation workflow, a specific antibody was produced and utilized to confirm the identified modifications. In this study, the acetylation at threonine 22 of human histone H3.3 (H3T22) was selected and the modified peptide KQLATacKAAR (H3T22ac) was chemically synthesized. Notably, H3T22 was a conserved site in human histone H3 variants including H3.1, H3.2, H3.3, H3t, H3.X and H3.Y . There were plenty of PTMs reported near H3T22 such as arginine 17 (R17), lysine 18 (K18), lysine 23 (K23) and lysine 27 Edoxaban (tosylate Monohydrate) (K27). The Edoxaban (tosylate Monohydrate) acetylation of K18 and K23 was found to regulate the activity of coactivator-associated arginine methyltransferase-1 (CARM1) to methylate R17 [31,32]. Recently, acetylation of H3S22 from and H3T22 from was reported . To confirm acetylation of human histone H3T22, a polyclonal antibody recognizing the acetylated human histone H3T22 (H3T22ac) was produced and affinity purified using the modified peptide KQLATacKAAR. The anti-H3T22ac antibody selectively recognized a peptide with the acetylated residue (H3T22ac), but did not Edoxaban (tosylate Monohydrate) detect the same peptide without the acetylated residue (H3T22), or different peptides with acetylated serine at different sites (Figure 4a), confirming the specificity of the antibody. Finally, the anti-H3T22ac antibody was subsequently used to detect the acetylated H3T22ac in HEK293T and HeLa cell lysates by western blot assay (Figure 4b). The anti-H3 antibody was used to show the presence and the position of human histone H3. Positive signals were detected at the same position by the anti-H3T22ac antibody, indicating the presence of the acetylated threonine 22 on human histone H3. Open in a separate window Figure 4 Validation of the acetyl-T22 in human histone H3 by immune assays. (a) Specificity of the anti-H3T22ac antibody was demonstrated by dot blot assay using four synthetic peptides: H3T22, un-acetylated peptide KQLATKAAR; H3T22ac, acetylated peptide KQLATacKAAR; M8S253ac, acetylated peptide KSacPLTEPNFENKC; M8S71ac, acetylated peptide TSacITPSSQDICRICHCEGDC. 50 and Edoxaban (tosylate Monohydrate) 500 ng Edoxaban (tosylate Monohydrate) of the peptides were used as indicated; (b) Detection of H3T22ac in human histones from different cell lysates. HEK293T (lane 1) and HeLa (lane 2) cell lysates were resolved by SDS-PAGE and detected by western blot assay using the antibodies against H3 (bottom) and H3T22ac (top). 4. Conclusions In this study, a wide tolerance acetylation workflow was used to identify new modifications mimicking the addition of 42 0.5 Da delta mass at undefined amino acid residues of human histones. Shotgun proteomics using liquid chromatographyCtandem mass spectrometry (LCCMS/MS) followed with a multi-blind spectral alignment algorithm using a wide delta mass tolerance of 42 0.5 Da modifications revealed a frequent occurrence of 42 0.5 Da modifications at serine, threonine, as well as lysine residues. Accurate delta mass clustering.