Quantitative Analysis of global Ubiquitination in HeLa Cells by Mass Spectrometry

Abstract

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.

The Lysine 48 and Lysine 63 Ubiquitin Conjugates Are Processed Differently by the 26 S Proteasome

“…In contrast, other detectable linkages including the Lys-6, -11, -27, -29, and -48 increased promptly. Under a severe proteasome inhibition condition (10 M MG132 for 15 h) we found that the cellular levels of all detectable Ub linkages (Lys-11, -48, and -63) increased (data not shown), consistent with two recent studies in yeast and mammalian cells (37,38). The increase of the cellular level of Lys-63 polyUbs in the later time of proteasome inhibition would suggest that most Lys-63 chains are not used as signals for proteasomal degradation; otherwise, they would have promptly increased after proteasome inhibition in a similar manner as the Lys-48 polyUbs.…”

Deubiquitylases From Genes to Organism

“…Proteomic studies reveal that each of the isopeptide linkage types is represented to a significant extent in both yeast and mammalian cells (54,117,196,282,298). The attachment of ubiquitin chains with any isopeptide linkage excepting Lys63 appears to target substrates to the proteasome in vivo (54,172). Lys63 linkages may be particularly important in targeting endosomal proteins to the lysosome (38, 142), whilst linear ubiquitin chains play a critical role in the NF B signaling pathway (269).…”

Protein Ubiquitination in IR-Induced DNA Damage Response

“…A similar approach was adapted in the mammalian system, in which a tandem His-biotin-tagged Ub was stably expressed in HeLa cells for efficient substrate isolation. In this experiment, ~ 50 ubiquitination sites were identified with a LTQOrbitrap mass spectrometer (213). Recently, the identification of ~750 lysine ubiquitination sites from two human cell lines that stably express HA-Ub was reported, which stands, by far, as the largest collection of ubiquitination sites (202).…”