Quantitative Analysis of the Sirt5-Regulated Lysine Succinylation Proteome in Mammalian Cells.

Lysine (Lys) succinylation is a recently discovered protein posttranslational modification pathway that is evolutionarily conserved from bacteria to mammals. It is regulated by Sirt5, a member of the class III histone deacetylases (HDACs) or the Sirtuins. Recent studies demonstrated that Lys succinylation and Sirt5 are involved in diverse cellular metabolic processes including urea cycle, ammonia transfer, and glucose metabolism. In this chapter, we describe the general protocol to identify Sirt5-regulated Lys succinylation substrates and a computational method to calculate the absolute modification stoichiometries of Lys succinylation sites. The strategy employs Stable Isotope Labeling of Amino acid in Cell culture (SILAC) and the immunoaffinity enrichment of Lys succinylated peptides to identify the Lys succinylation sites that are significantly upregulated in Sirt5 knockout mouse embryonic fibroblast cells.