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Ubiquitination, SILAC, HB-tag, Tandem-affinity purification, Ubiquitin-chain topology, Mass spectrometry
Abstract:
The covalent attachment of the small protein ubiquitin to other proteins is known to control a host of
biological pathways and is emerging as an important regulatory factor in various processes specific to the
nervous system. Ubiquitination is also tightly linked to most neurodegenerative disorders. A quantitative,
proteome-wide view of the dynamic changes in ubiquitin modification associated with neuronal
activity states and various stages of neurodegenerative disorders is therefore desired. Advances in quantitative
mass spectrometry and the development of new biological tools make these approaches feasible for
many laboratories. We describe here a combination of SILAC-based (stable isotope labeling by amino
acids in cell culture) quantitative mass spectrometry and tandem-affinity purification to detect systemwide
changes in ubiquitination and ubiquitin chain topologies that will be useful to probe the role of
ubiquitin in the nervous system.
