Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry.
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.
Meierhofer
David
D
Department of Biological Chemistry, University of California, Irvine, California 92697, USA.
Wang
Xiaorong
X
Huang
Lan
L
Kaiser
Peter
P
D000595:Amino Acid Sequence; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D007976:Leupeptins / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism
D007976:Leupeptins; D010455:Peptides; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D005982:Glutathione Transferase; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
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