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.
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Christopher M
CM
Rose
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Marta
M
Isasa
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Alban
A
Ordureau
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Miguel A
MA
Prado
Cell Signaling Technology, Danvers, MA 01923, USA.
Sean A
SA
Beausoleil
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Mark P
MP
Jedrychowski
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Daniel J
DJ
Finley
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
J Wade
JW
Harper
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: steven_gygi@hms.harvard.edu.
Steven P
SP
Gygi
D000818:Animals; D001343:Autophagy; D013058:Mass Spectrometry; D008928:Mitochondria; D063306:Mitophagy; D010300:Parkinson Disease; D011494:Protein Kinases; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination
D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases
isobaric labeling; mitochondria; mitophagy; post-translational modification; proteasome; proteomics; quantitative mass spectrometry; systems biology; ubiquitylation
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