Dataset Information

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

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Anita Saraf

PROVIDER: MSV000084419 | MassIVE | Fri Oct 04 10:58:00 BST 2019

SECONDARY ACCESSION(S): PXD015705

REPOSITORIES: MassIVE

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Publications

NRBP1-Containing CRL2/CRL4A Regulates Amyloid β Production by Targeting BRI2 and BRI3 for Degradation.

Yasukawa Takashi T Tsutsui Aya A Tomomori-Sato Chieri C Sato Shigeo S Saraf Anita A Washburn Michael P MP Florens Laurence L Terada Tohru T Shimizu Kentaro K Conaway Ronald C RC Conaway Joan W JW Aso Teijiro T

Cell reports 20200301 10

Alzheimer's disease (AD) is a progressive neurodegenerative disease caused by accumulations of Aβ peptides. Production and fibrillation of Aβ are downregulated by BRI2 and BRI3, which are physiological inhibitors of amyloid precursor protein (APP) processing and Aβ oligomerization. Here, we identify nuclear receptor binding protein 1 (NRBP1) as a substrate receptor of a Cullin-RING ubiquitin ligase (CRL) that targets BRI2 and BRI3 for degradation. Moreover, we demonstrate that (1) dimerized NRBP ...[more]

PMID: 32160551

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