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. 2012 Apr 2;109(16):E916-E925. doi: 10.1073/pnas.1121471109

Fig. P1.

Fig. P1.

Diagram of the biotinyl-lysine method and its application to identifying histone H3 K79 as a Sir2 substrate. Free lysines (K) in proteins are chemically acetylated (Ac) with acetic anhydride, and the protein is incubated with a sirtuin and NAD+ to deacetylate selected lysine residues. The deacetylated lysines are conjugated with biotin (B), and then mass spectrometry methods (MALDI-TOF or LC ESI MS/MS) are used to identify the biotinylated sites. This method was utilized to identify a site in histone H3, K79, that is acetylated at low levels in vivo and is deacetylated by Sir2.