Global ubiquitination analysis by SILAC in mammalian cells

Ubiquitination is a versatile and dynamic posttranslational modification in cells, regulating almost all cellular events. With rapid developments of affinity capture reagents and high-resolution mass spectrometry, it is now feasible to globally analyze the ubiquitinated proteome (ubiquitome) using quantitative strategies, such as stable isotope labeling with amino acids in cell culture (SILAC). Here we describe in detail a SILAC protocol to profile the ubiquitome in mammalian cells including protein labeling, antibody-based enrichment, and analysis by mass spectrometry.