Identification and Quantification of K63-Ubiquitinated Proteins in Neuronal Cells by High-Resolution Mass Spectrometry
Protein ubiquitination is a widespread modification serving many roles in neuronal development and function. Moreover, the accumulation of ubiquitinated proteins is a prominent feature of neurodegeneration and oxidative stress related diseases. The emerging diversity of ubiquitin signals beyond protein degradation—based on distinct types of polyubiquitin chains—necessitates tools that specifically and quantitatively investigate its different functions. Polyubiquitin chains linked by lysine 63 (K63) relate to neurodegenerative diseases, but most of their targets and functions have not yet been elucidated. K63-linked ubiquitin has been implicated in DNA repair signaling, endocytosis, and inclusion body clearance. In addition, we recently identified an important role of K63 ubiquitin in regulating translation in response to oxidative stress in yeast. The change in K63 ubiquitination in response to hydrogen peroxide is conserved in mouse hippocampal HT22 cells, highlighting the importance of this modification in higher eukaryotes. In this chapter, we discuss cutting-edge methodologies available to investigate protein ubiquitination in a proteome-wide and quantitative manner, and we present a method to simultaneously isolate and identify the specific targets of K63 ubiquitin. This method relies on the use of a selective K63 ubiquitin isolation tool with subsequent analysis of protein content by high-resolution mass spectrometry. The proposed workflow can be combined with additional methods for ubiquitin analysis and applied to several research models. This approach can also provide the scientific basis for the development of new tools to isolate and identify targets of other ubiquitin linkages.
Keywords:K63 ubiquitin Mass spectrometry Neurons Oxidative stress
This work was supported in part by the US National Science Foundation EAGER grant MCB-1355462 (CV), by the US National Institutes of Health K99 award ES025835 (GMS), and the Zegar Family Foundation Fund for Genomics Research at New York University (CV).
We thank R. Ratan (Burke Medical Research Institute) for providing the HT22 cells.