Datasets from an interaction proteomics screen for substrates of the SCF(βTrCP) ubiquitin ligase.

Roberto Magliozzi,Albert J.R Heck,Mao Peng,Daniele Guardavaccaro,Shabaz Mohammed,Teck Yew Low

doi:10.1016/j.dib.2015.05.003

Abstract

An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCFβTrCP and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T.Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R. Heck, A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase. Sci. Signal. 7 (2014) rs8–rs8, 10.1126/scisignal.2005882.

Highlights

An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase
A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCFβTrCP and unspecific binders
Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry

Summary

Experimental design

SCFβTrCP is a multi-subunit E3 ubiquitin ligase consisting of Cul, Rbx, Skp, and the F-box protein βTrCP. I.e. without any βTrCP2-based fusion protein (EV), were used as negative control. The first mutant, βTrCP2-R447A, harbors an Arg to Ala mutation at position 447 of βTrCP2. This substitution within the WD40 β-propeller domain abrogates the interaction with and the ubiquitylation of SCFβTrCP substrates [3,4]. The second mutant, βTrCP2-ΔF, carries a deletion in the F-box motif and acts as a dominant negative mutant [5,6] since it binds substrates but is unable to interact with Skp, Cul, Rbx and the E2 enzyme

Materials and methods

DNA constructs

Recovery of immunoprecipitated proteins for LC–MSMS

LC–MSMS

LC–MSMS data analysis

Post-acquisition data analysis CRAPome and Perseus analysis