Dataset Information

Detection of Cas9 post-translational modifications by mass spectrometry

ABSTRACT: We aimed to detect potential ubiquitylation of Cas9 expressed in human cells by mass spectrometry

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Frank Stein

LAB HEAD: Umut Şahin

PROVIDER: PXD025062 | Pride | 2022-01-11

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	F083953.dat	Other
	F083955.dat	Other
	F098864.dat	Other
	Lara_190618_P0991_MR_US_CUB_1out10_HCD_60min_R1.raw	Raw
	Lara_190618_P0991_MR_US_CUB_MG_1out10_HCD_60min_R1.raw	Raw

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Project description:Ubiquitination of proteins is central to protein homeostasis and other cellular processes including DNA repair, vesicular transport, cell-division etc. The process of ubiquitination is conserved from yeast to humans and is carried out by the sequential action of three enzymes: E1, E2 and E3. There are an estimated >600 E3 ligases in humans that execute ubiquitination of specific target proteins in a spatio-temporal manner to elicit desired signaling effects. Here, we developed a simple, proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. Our method exploits the proximity and the relative orientation of the E3-ligase catalytic domain with respect to ubiquitin observed in the enzymatic intermediate-state structures of E3-E2~Ub. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich the bona fide substrates and potential new substrates of the ligase using a one-step Streptavidin pulldown under denaturing conditions. As proof-of-principle, we applied our method, which we named Ub-POD, to the RING E3 ligase Rad18. Rad18 ubiquitinates DNA-sliding clamp PCNA upon UV-induced DNA damage. We identified PCNA and several other critical players in the DNA damage repair pathway in a single Ub-POD experiment. We further discovered an unintended but important application of Ub-POD, where we were able to pin down the cellular localization of Rad18- mediated ubiquitination to the damaged DNA nuclear puncta through Streptavidin immunofluorescence. We also applied our method to TRAF6, another RING ubiquitin ligase involved in TNF signaling, successfully identifying known and potentially new substrates. Finally, we adapted our method to the U-box type E3 ubiquitin ligase CHIP to demonstrate that we can identify substrates of two major classes of mammalian ubiquitin ligases. We anticipate that our method and principle could be widely adapted to all classes of ubiquitin ligases to identify substrates and localize the cellular site(s) of ubiquitination.

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Detection of Cas9 post-translational modifications by mass spectrometry

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