Figure 7.

Discovery of naturally occurring Kme₂ proteins and Kme₂-sites from a complex cell lysate was carried out by TACO chemistry. (a) Selective modification of naturally occurring dimethyl lysine in nucleosomes using TACO chemistry followed by the enrichment of Kme₂-generated aldehyde proteins using hydroxylamine resin and release of trapped proteins under acidic conditions. The filtrate contains proteins remaining after the enrichment process with and without TACO chemistry on nucleosomes, while the eluate comprises proteins enriched from the nucleosomes with and without TACO chemistry using a solid support. The filtrate and eluate were analyzed by SDS PAGE analysis. The gels showed the enrichment of Kme₂ proteins from nucleosomes under TACO reaction conditions. (b) Selective modification of Kme₂ proteins by TACO chemistry followed by trapping of Kme₂-generated aldehyde proteins by reductive amination and proteomic analysis. Twelve novel Kme₂ proteins and thirteen new Kme₂ sites were identified (novel = blue, known = red). In the negative control, five proteins were identified, which vary from the TACO-treated samples due to the presence of natural occurring allysine on proteins. (c) Enrichment of TACO-modified Kme₂ proteins followed by proteomic analysis. Nine low-abundance Kme₂ proteins were discovered with nine new Kme₂ sites.