Comparative SUMO Proteome Analysis Using Stable Isotopic Labeling by Amino Acids (SILAC).

ABSTRACT

Sumoylation is a dynamic protein posttranslational modification that contributes to many intracellular pathways, including nucleocytoplasmic transport, DNA repair, transcriptional control, and chromatin remodeling. Interestingly, various stress conditions such as heat shock, oxidative stress, and ischemia promote global changes in sumoylation in different cells or tissues. However, due to limitations in either abundance or steady state sumoylation level, it is often difficult to detect differences in the sumoylation of a protein under different conditions simply by immunoblotting. In the last decade, the enrichment of endogenous sumoylated proteins has been greatly improved using immunoprecipitation techniques. Combining these methods with quantitative methodologies such as Stable Isotopic Labeling with Amino Acids in Cell culture (SILAC), it is feasible to identify the sumoylation status of a wide range of proteins and detect changes in SUMO conjugation under different experimental conditions. In this chapter, we describe a method that allows comparison of the sumoylated proteome in HeLa cells between two conditions, using differential labeling by light or heavy amino acids (SILAC), isolation of endogenous sumoylated (SUMO1 and SUMO2/3) proteins with immunoprecipitation and MS analysis. We also discuss the conceptual design and the considerations before performing such an experiment.