Global Analysis of Ubiquitination
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 system-wide changes in ubiquitination and ubiquitin chain topologies that will be useful to probe the role of ubiquitin in the nervous system.
Key wordsUbiquitination SILAC HB-tag Tandem-affinity purification Ubiquitin-chain topology Mass spectrometry
This work was supported by NIH (GM66164 and CA113823). David Meierhofer was an Erwin Schrödinger fellow supported by the FWF Austria.
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