MassIVE MSV000084419

Complete Public PXD015705

Mudit analyses of the proteins substrates candidate of NRBP1 using TR-TUBE system

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Description

293 stable cell line expressing NRBP1 or NRBP1 mutant were transfected with HA-TR-TUBE using TR-TUBE system to protect the degradation of the ubiquitin chains of the potential ligase substrate. The complexes were pulled down using anti-HA antibody and subsequently digested with modified Trypsin. Anti-diGly Antibody was used for isolation of ubiquitinated peptides which recognizes the Gly-Gly dipeptide bond attached to lysine. Enriched peptide pellets were dissolved in 100 micro liter of buffer A, 5 percent acetonitrile, 0.1 percent formic acid. Samples were pressure loaded on split-triple-phase fused-silica micro-capillary columns and analyzed by nanoflow liquid chromatography using an ultimate 3000 RSLC nano system coupled to a Q Exactive plus mass spectrometer equipped with a nanospray Flex Ion source, Thermo Fisher Scientific and analyzed using a 10-step MudPIT analysis. The MS-MS datasets were searched using ProLuCID. The samples were searched against a database of 146186 sequences, consisting of 72956 H. sapiens non-redundant proteins NCBI, 2015-03-25, 193 usual contaminants -such as human keratins, IgGs, and proteolytic enzymes, and the NRBP1-muant protein sequences. To estimate false discovery rates, each protein sequence was randomized leading to a total search space of 73091 sequences. The precursor and fragment mass tolerances were set to 10 ppm and 100 ppm, respectively. Diglycine modification of Lys side chains, Ser-Thr-Tyr phosphorylation, pyroglutamate formation, acetylation, methionine oxidation and cysteine methylthio were set as variable modifications. Peptide-spectrum matches were sorted, selected and compared using DTASelect together with in-house software swallow and sandmartin. Proteins had to be detected by at least 2 spectra and average FDRs at the protein and spectral levels were set to less than 4 percent. To estimate relative protein levels, Normalized Spectral Abundance Factors,dNSAFs, were calculated for each detected protein. NSAF7 was used to extract total and modified label-free features for each amino acid within and calculate modification levels based on local spectral counts and generate the modification results. [doi:10.25345/C5PM32] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: E3 ubiquitin ligase ; ubiquitination ; protein degradation

Contact

Principal Investigators:
(in alphabetical order) 		Anita Saraf, The Stowers Institute for Medical Research, United States
Submitting User: asaraf

Publications

Yasukawa T, Tsutsui A, Tomomori-Sato C, Sato S, Saraf A, Washburn MP, Florens L, Terada T, Shimizu K, Conaway RC, Conaway JW, Aso T.
NRBP1-Containing CRL2/CRL4A Regulates Amyloid ? Production by Targeting BRI2 and BRI3 for Degradation.
Cell Rep. 2020 Mar 10;30(10):3478-3491.e6.

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