Highly Multiplexed Quantitative Mass Spectrometry Analysis of Ubiquitylomes

System-wide quantitative analysis of ubiquitylomes has proven to be a valuable tool for elucidating targets and mechanisms of the ubiquitin-driven signaling systems, as well as gaining insights into neurodegenerative diseases and cancer. Current mass spectrometry methods for ubiquitylome detection require large amounts of starting material and rely on stochastic data collection to increase replicate analyses. We describe a method compatible with cell line and tissue samples for large-scale quantification of 5,000–9,000 ubiquitylation forms across ten samples simultaneously. Using this method, we reveal site-specific ubiquitylation in mammalian brain and liver tissues, as well as in cancer cells undergoing proteasome inhibition. To demonstrate the power of the approach for signal-dependent ubiquitylation, we examined protein and ubiquitylation dynamics for mitochondria undergoing PARKIN- and PINK1-dependent mitophagy. This analysis revealed the largest collection of PARKIN- and PINK1-dependent ubiquitylation targets to date in a single experiment, and it also revealed a subset of proteins recruited to the mitochondria during mitophagy. [Display omitted] •Reproducible, accurate method for quantitative measurement of ubiquitylation sites•Analysis of 5,000–9,000 ubiquitylation forms across ten tissue or cell culture samples•Site-specific ubiquitylation of cancer-related proteins upon bortezomib treatment•PINK1- and PARKIN-dependent ubiquitylation events during early and late mitophagy Large-scale simultaneous ubiquitylome analysis of ten cell line or tissue samples results in the quantitation of 5,000–9,000 ubiquitylation forms, and it reveals PINK1- and PARKIN-dependent ubiquitylation events during early and late mitophagy.